The First Fifteen Years of Steroid Receptor Research in Zebrafish; Characterization and Functional Analysis of the Receptors

Steroid hormones regulate a wide range of processes in our body, and their effects are mediated by steroid receptors. In addition to their physiological role, these receptors mediate the effects of endocrine disrupting chemicals (EDCs) and are widely used targets for dugs involved in the treatment of numerous diseases, ranging from cancer to inflammatory disorders. Over the last fifteen years, the zebrafish has increasingly been used as an animal model in steroid receptor research. Orthologues of all human steroid receptor genes appear to be present in zebrafish. All zebrafish steroid receptors have been characterized in detail, and their expression patterns have been analyzed. Functional studies have been performed using morpholino knockdown of receptor expression and zebrafish lines carrying mutations in one of their steroid receptor genes. To investigate the activity of the receptors in vivo, specific zebrafish reporter lines have been developed, and transcriptomic studies have been carried out to identify biomarkers for steroid receptor action. In this review, an overview of research on steroid receptors in zebrafish is presented, and it is concluded that further exploitation of the possibilities of the zebrafish model system will contribute significantly to the advancement of steroid receptor research in the next decade.

Steroids and Steroid Receptors
Steroid hormones are signaling molecules that regulate a wide variety of physiological processes in our body. Two main classes of steroid hormones exist: sex steroids and corticosteroids. The secretion of sex steroids is controlled by the hypothalamus-pituitary-gonadal axis, and these hormones are primarily produced in the ovary in females and in the testis in males. Sex steroids regulate sexual differentiation and reproduction, and they can be subdivided into three groups: estrogens, progestogens and androgens. The hypothalamus-pituitary-adrenal axis regulates the secretion of corticosteroids, which are synthesized in the cortex of the adrenal gland. They can be subdivided into two groups: mineralocorticoids, which regulate our salt and water balance, and glucocorticoids, which are involved in the stress response and regulate processes like glucose metabolism and immune function.
All steroid hormones are synthesized from the common precursor molecule cholesterol. The rate-limiting step in this process is the transport of cholesterol across the mitochondrial membranes, which is controlled by steroidogenic acute regulatory protein (StAR). Next, cholesterol is converted to pregnenolone, which is subsequently converted to progesterone, the main endogenous progestogen in our body. From progesterone, the mineralocorticoid aldosterone is Steroid receptors are located intracellularly, and in the absence of hormone they form a multiprotein complex with heat shock proteins and immunophilins, which keep them in an inactive state with high affinity for their cognate ligands. Upon ligand binding, the receptors dissociate from the repressor protein complex and a conformational change is induced. In the active conformation, the receptors act as transcription factors. They are able to bind to specific target sites in the DNA, called hormone response elements (HREs), and upon recruitment of different types of transcriptional coregulator proteins the transcriptional rate of a nearby gene is altered. Additionally, steroid receptors are able to alter gene transcription by interacting with other transcription factors and alter the activity of these proteins. Besides these 'genomic' actions of steroid receptors, rapid 'nongenomic' mechanisms of steroid receptor action have been demonstrated, but the intracellular signaling pathways underlying these effects have not been unraveled in full detail yet. Finally, the G protein-coupled estrogen receptor (GPER, previously termed GPR30) has been shown to mediate rapid effects of estradiol and aldosterone [2], and progesterone is known to activate membrane Progestin Receptors (mPRs), which are members of the Progestin and AdipoQ Receptor family [3]. These latter two receptor types are beyond the scope of this review, which will focus on the classical steroid receptors from the nuclear receptor superfamily.

Steroid Receptor Research in Zebrafish
In this review, an overview is presented of research on steroid receptors using the zebrafish (Danio rerio) as a model system. The zebrafish was originally used as an animal model for studies on embryonic development and morphogenesis, and it is still widely used in research on vertebrate development [4,5]. However, over the last decade the zebrafish has also emerged as an important model system for eco-toxicological [6] and biomedical research [7,8]. Several characteristics make it a highly versatile research model. Their easy maintenance, high fecundity and the small size and optical transparency of the embryos and larvae make them highly suitable A large amount of the research that has been performed on steroid receptors in zebrafish has been aimed at studying the effects of endocrine disrupting chemicals (EDCs). EDCs are natural or synthetic compounds that occur in the environment and disrupt the function, levels and distribution of endogenous hormones of exposed organisms, by mimicking or antagonizing the actions of hormones, or by modulating hormone synthesis and metabolism [9]. This may ultimately lead to altered development and/or reproduction in humans and wildlife. EDCs form a heterogeneous group of substances, encompassing natural and synthetic hormones and seemingly unrelated compounds like pesticides, polychlorinated biphenyls (PCBs), Bisphenol A (BPA), phthalates, flavonoids and polycyclic musks [10,11]. The main mechanism of action for these compounds is generally suggested to be agonistic or antagonistic interaction with steroid receptors. Zebrafish are commonly used for monitoring EDC activity. Phenotypic endpoints like growth, sexual differentiation, sex ratio, egg production and fertilization success are commonly used for monitoring EDC activity, and these biomarkers are used in partial and full life cycle studies [12,13]. A lot of studies on zebrafish steroid receptor action have aimed to identify novel molecular biomarkers or screening approaches for EDC activity which could replace or complement the phenotypic endpoints. In addition, many investigations were focused on unraveling the mechanism of action of EDCs, especially using approaches for genetic knockdown of steroid receptor function.
Research on steroid receptors in zebrafish started in 2002 with the characterization of the zebrafish ERs, and almost ten years later all zebrafish steroid receptors had been identified and characterized (for an overview see Table 1). All mammalian steroid receptor genes appeared to have one orthologue in zebrafish, apart from the gene encoding Er , of which two copies were found in the zebrafish genome. As a first step in the characterization, the zebrafish receptors were used in reporter assays in cultured (mostly mammalian) cells. In general, the ligand specificity did not differ much from the mammalian receptors. Second, expression analysis was performed, mostly by in situ hybridization, since the availability of antibodies specific for (zebra)fish receptors is limited. After these initial steps, functional in-vivo studies have been performed. Knockdown of the receptor using morpholinos, which are antisense oligonucleotide analogs often used in zebrafish research, has revealed the function of the receptor during embryonic and larval stages. In recent years, it has become clear that some of the phenotypes caused by this approach may result from unintended nonspecifc morpholino actions [14]. This issue may be overcome by comparing the results from morpholino studies to data obtained using zebrafish lines with mutations in steroid receptor genes, and this has provided further insight into the in vivo action of several steroid receptors. For Er and Gr, luciferase and Green Fluorescent Protein (GFP) reporter lines have been generated which allow in-vivo monitoring of the activity of these receptors. Many functional studies have used transcriptomic analysis, initially using microarray and more recently using RNA sequencing, to elucidate molecular pathways regulated by steroid receptor action and to identify biomarkers for steroid receptor activity. In the following sections,  for each of the five steroid receptor types the zebrafish research performed over the last fifteen years will be discussed.

The Zebrafish Ers
Fifteen years ago, several groups reported on the identification of three different Er encoding genes in zebrafish [15][16][17]. Phylogenetic analysis showed that one cDNA originated from the zebrafish orthologue of the mammalian ER gene, and that the other two cDNAs were products from two ER orthologues. It was concluded that the identified er cDNAs originated from duplicated er genes in the zebrafish genomes. These duplicate er genes had been shown previously in other fish species like goldfish [18] and atlantic croaker [19]. Er 2 had a slightly higher affinity for E2 than the other two zebrafish Ers, which was reflected in a higher potency in in-vitro reporter assays, in which a luciferase gene was driven by a promoter containing an estrogen response element (ERE) [15,20]. E2, the metabolites estrone (E1) and estriol (E3), and the synthetic estrogen diethylstilbestrol (DES) showed similar agonistic activity on all three receptors [15,16], whereas the synthetic ligands ICI 164384 (ICI) and 4-hydroxytamoxifen (OHT) acted as antagonists on all zebrafish Ers. Expression analysis of the three genes encoding Ers showed that during zebrafish development, three phases can be distinguished [16,20,21]. During the first phase, the presence of maternal transcripts for Er 1 [20][21][22] and Er 2 [16] has been demonstrated by RT-PCR and RNAse protection assay respectively. Between the mid-blastula stage and 72 hours post fertilization (hpf), the expression levels of all three er genes is decreased, although by in-situ hybridization it was shown that some cell types show high levels of specific er mRNAs. For example, epidermal cells were demonstrated to contain high er 2 mRNA levels at this stage [21]. After 72 hpf, all er genes start to be expressed at high levels [16,[20][21][22], and particularly high levels of er 1 and er 2 expression were found during the second week of development in neuromasts of the lateral line, a mechanoreceptive system specific to aquatic vertebrates [21]. In adult fish, expression of all three receptor subtypes was demonstrated by RT-PCR in tissues involved in reproduction like brain, pituitary, ovary and testis, but also in nonreproductive tissues like liver, intestine and eyes [15,22,23]. In-situ hybridization on brains of adult females showed distinct but partially overlapping patterns of expression in two neuroendocrine regions, doi:10.11131/2017/101286 the preoptic area and the mediobasal hypothalamus [15]. Hepatic expression of er mRNA in adult fish was upregulated upon E2 treatment, and this effect appeared to be mediated by Er and Er 2, but not Er 1 [23,24].
Knockdown of the expression of the zebrafish er genes has been performed in several studies. Temporary knockdown of the er 2 expression using a morpholino resulted in the disrupted development of the neuromasts (absence of hair cells) in the morpholino-treated embryos, which was attributed to an aberrant activation of the Notch signaling pathway [25]. Induction of specific target genes of Er 2 and Er could also be inhibited by morpholino knockdown of the receptor [26]. Knockdown of er expression during embryonic stages by morpholino-induced blocking of translation of maternal mRNA resulted in severe developmental defects and early mortality [27]. Using a combination of chemical mutagenesis and high-throughput sequencing [28], a mutant zebrafish line was generated, which carried a mutation in the gene encoding Er 2 (resulting in an 8 amino acid insertion in the ligand-binding domain [29]). Unfortunately, the readouts used in studies on this mutant line were different from those used in the morpholino studies, so validation of the morpholino data was not possible. This mutant line displayed increased testosterone and 17 -estradiol (E2) levels and, probably as a result of the hormonal imbalance, distorted sexual ratios and altered testicular morphology. It was concluded that Er 2 plays an important role in the feedback regulation of steroid biosynthesis, in line with its elevated expression in the gonads and neuroendocrine regions of the brain. In addition, a decreased immune response to a viral infection was found in this mutant fish [29].
Several transgenic reporter fish lines are available to study the activity of Er in vivo. In these lines, an Er-responsive promoter is genetically fused to a reporter gene encoding either luciferase or GFP. Using a transgenic line with the luciferase gene coupled to a promoter with a single estrogen response element (ERE), Er activity could be measured after 96 hour exposure to estrogens by measuring the luciferase activity in tissue homogenates [17,30]. This assay has been performed using adult zebrafish and juveniles, in which adults appear to be more responsive. In addition, two transgenic zebrafish lines have been generated in which GFP is used, which makes it possible to locate the reporter activity using fluorescence microscopy. In the first line, a promoter with 5 EREs was utilized, and GFP expression was observed in embryonic and larval zebrafish in the brain, liver and pancreas after estrogen exposure [31,32]. Estrogen treatment of larvae also induced expression of GFP in cells in the ventral fin adjacent to the cloaca, in the developing olfactory organ, and in the heart. This induction could be inhibited by the ER antagonist ICI182,780 (fulvestrant). Interestingly, GFP induction by estrogens like EE2 and genistein showed marked differences in the tissue specificity of the response [31]. In adult fish, males showed no GFP expression in the liver, whereas females displayed dim expression in this tissue, which could be increased upon estrogen exposure. Ovaries and (male and female) pituitaries were GFP-positive without exposure to exogenous estrogens, so this was considered to reflect Er activation by endogenous estradiol. In another transgenic line GFP was coupled to the promoter of the brain aromatase b gene (cyp19a1b), resulting in specific GFP expression in radial glial cells in the area bordering the brain ventricles [33]. Before 9 days post fertilization (dpf), the GFP expression was only observed after induction by estrogens [34].
Various transcriptomic analyses have been carried out in order to identify novel molecular biomarkers for Er activity [35]. The liver and telencephalon transcriptome have been determined using microarray analysis of adult male and female fish after exposure to EE2 [36,37]. Whole doi:10.11131/2017/101286 body transcriptome analysis was performed on male adult fish after exposure to E2 in another microarray study [38]. Microarray analysis has also been performed during early developmental stages (1, 2, 3 and 4 dpf). In addition to well-known Er target genes like vtg1 (the only gene induced by estrogens at all stages studied [32]), vtg3, vtg4 and er , these studies also revealed Er regulation of many metabolic genes [36,37] and genes involved in cell cycle and DNA repair [38].

The Zebrafish Pr
Just like in most other teleost fish species, a single pr gene was found in zebrafish [39,40], although duplicate pr genes (pr1 and pr2) have been demonstrated in the European eel [41]. The two pr genes in the eel, like many other duplicated genes, result from a whole genome duplication that happened at the base of teleost evolution around 320-350 million years ago [42]. The orthologue of the eel pr2 gene was most likely lost early during teleost evolution, soon after the order of Anguilliformes (to which the eels belongs) had branched off the main teleost lineage. The zebrafish Pr binds with high affinity (k in the nanomolar range) to the main endogenous progestogen in zebrafish, 17 ,20 -dihydroxy-4-pregnen-3-one (DHP) [39]. DHP is converted from 17-OH-progesterone by 20 -hydroxysteroid dehydrogenase (Hsd20b), an enzyme that has only been identified in teleosts [43,44]. The zebrafish Pr activated the mouse mammary tumor virus (MMTV) promoter in luciferase assays upon binding to DHP, progesterone, 17-OH-progesterone, 17 ,20 ,21-trihydroxy-4-pregnen-3-one (20 -S, another fishspecific endogenous progestogen), and the synthetic progestin promegestone (R5020) [39,40]. In testicular explants, the release of 11-KT was induced by DHP and this response could be inhibited by the antagonist RU486 [40].
Pr expression analysis using RT-PCR showed no maternally deposited transcripts, and the first detectable mRNA levels were found at 8 hpf [40]. In adults, the pr transcript and protein were detected by RT-PCR and western blot and appeared to be abundant in the ovaries, testis, and brain and scarce or undetectable in the liver, intestine, muscle, heart and gills [39,40]. In the ovary, high mRNA and protein levels were found in follicular cells and early stage oocytes (stages I and II), with very low levels within maturationally competent late-stage oocytes (IV), which is in line with the nondetectable levels in early-stage fertilized embryos [39,45]. Similarly, in the testis, mRNA and protein expression was observed in spermatogonia and early spermatocytes and not in spermatids or sperm [39]. In the brain, immunohistochemical analysis showed robust Pr levels in the two main neuroendocrine regions of the brain: the preoptic area and the mediobasal hypothalamus [39]. These expression data indicate that Pr regulates reproductive signaling in the brain, early germ cell proliferation in testis and ovarian follicular functions, but not final oocyte or sperm maturation.
Using transcription activator-like effector nucleases (TALENs), several pr mutant fish lines have been made [46,47]. Three lines were generated with small deletions in exon 1 of the gene encoding Pr, resulting in frameshifts in the gene starting at the sequence encoding amino acid 51, 47 and 205 [46]. Another pr mutant line was made by deletion of a large part (13.4 kb) of the pr gene between exon 1 and 6, resulting in a frame shift from the codon encoding amino acid 151 [45]. These mutants showed highly similar phenotypes. Homozygous females were infertile, whereas males were fertile [45,46]. In the mutant females, oocytes appeared to mature normally, doi:10.11131/2017/101286 Page 6 but these mature oocytes were trapped within the follicular cells and failed to ovulate from the ovaries, even after induction by human Chorionic Gonadotropin (hCG) [45,46]. Apparently, Pr activity is required for ovulation, and it was shown that the Prostaglandin E Receptor 4 was involved, since the expression of this receptor is induced upon Pr activation and antagonists of this receptor inhibited hCG-induced ovulation [45]. Transcriptomic analysis of follicular cells from pr mutant fish using RNA sequencing revealed gene networks involved in the induction of ovulation, that are well conserved between zebrafish, mice and humans, and in which genes are enriched that are involved in inflammation, apoptosis, vascularization, cell-matrix adhesion, extracellular matrix remodeling, and the cell cycle [47].

The Zebrafish Ar
The cloning and characterization of the zebrafish ar gene was reported by several groups [48][49][50]. Although in several other fish species the occurrence of duplicate ar genes had been reported, only one ar gene was identified in the zebrafish genome. A single copy of the ar gene is present in Cypriniformes (e.g. common carp, goldfish, zebrafish), Characiformes (e.g. black widow tetras) and Siluriformes (e.g. sharptooth catfish), so the duplicate copy must have been lost during the early evolution of the Otophysi lineage, which gave rise to these orders of fish species [51]. The zebrafish ar gene appears to be the orthologue of the gene that encodes the Ar-A isoform in fish species containing two ar genes.
The zebrafish Ar was demonstrated to bind the endogenous steroids T, DHT, 11-ketotestosterone (11-KT) with high affinity (k in nanomolar range), A2 with an approximately ten-fold lower affinity and the synthetic steroids 17 -methyltestosterone (MT) and 17 -dimethyl-19-nortestosterone (mibolerone, MB) with a slightly higher affinity [48,50]. Transcriptional activation of an MMTV promoter driving a luciferase gene in human and zebrafish cells was found to be similar for T, 11-KT, MT, and DHT (although for DHT the potency was lower in zebrafish cells than in human cells) [49,50]. Flutamide was shown to act as an antagonist of the zebrafish Ar in these reporter assays [50]. Additionally, It was demonstrated that the main endogenous Ar ligand in zebrafish is 11-KT, which is produced from androstenedione in a two-step process [50]. The biosynthesis of the main mammalian androgens, T and DHT, is limited in zebrafish [50,52], just like in other fish species [44].
Expression levels of the ar gene during development were determined using RT-PCR. Maternal ar mRNA was detected and the concentration of ar transcripts decreased until around 12 hpf, after which it shows a gradual increase until at least 14 dpf [49]. Using in-situ hybridization, ar transcripts were found in the presumptive pronephros and in olfactory placodes in 24 hpf embryos and at 3-5 dpf in the pineal organ anlage and the retina [53]. In adult fish, ar mRNA was detected in the gonads, brain, kidney, liver, skin, muscle and eye tissue by RT-PCR [49]. By insitu hybridization the presence of ar mRNA was demonstrated in the testis, in the subpopulation of Sertoli cells contacting early spermatogonia (De Waal 2008) and in brain regions known to be involved in neuroendocrine regulation, like the preoptic area, the periventricular hypothalamus and discrete telencephalic regions [53].
Microarray analysis of the transcriptome of livers from adult female zebrafish exposed to the androgen 17 -methyldihydrotestosterone (MDHT) revealed regulation of genes involved in steroid and retinoic acid metabolism, hormone transport and regulation of cell growth and doi:10.11131/2017/101286 proliferation [54]. In another study, the effect of exposure of adult fish to the AR antagonists flutamide and vinclozolin on the gonadal transcriptome in males and females was determined using microarray analysis [55]. Multiple pathways were shown to be regulated, which were involved in steroidogenesis, spermatogenesis and fertilization. These pathways included the integrin, actin, steroidogenic factor-1, Fgf receptor signaling pathway and the polyamine and androgen synthesis pathway [55]. The transcriptional effect of 11-KT exposure of zebrafish larvae between 5 and 6 dpf was investigated by whole body microarray analysis [56]. Four regulated genes involved in hormone metabolism were additionally studied in detail: cyp2k22, slco1f4, lipca, sult2st3, and it was demonstrated using the antagonist nilutamide that these genes are specific targets of Ar [56].

The Zebrafish Gr
Soon after the Ar, the zebrafish Gr was characterized [57]. Only a single gr gene was identified in the zebrafish genome, although many fish species were known to have two gr orthologues. Just like the other duplicate steroid receptor gene copies, the two gr orthologues were a result of the whole genome duplication that had occurred early in the teleost lineage, and they are called gr1 and gr2 [58]. Phylogenetic and syntenic analysis showed that the zebrafish genome contains an orthologue of the gr2 gene, and that an ancestor of the zebrafish has lost its gr1 gene due to a genomic rearrangement in chromosome 21 [57]. This loss must have been a relatively recent event, since the common carp, which belongs to the same family (Cyprinidae) as the zebrafish, has both gr1 and gr2 orthologue genes [58]. Luciferase assays showed that the zebrafish Gr is able to activate the MMTV promoter upon binding to the synthetic glucocorticoid dexamethasone (EC 50 in the subnanomolar range) and the endogenous hormone cortisol (with a ∼30-fold lower potency) [57]. However, dexamethasone and cortisol were inactive in a larval regeneration-inhibition assay in which other glucocorticoids like beclomethasone dipropionate were shown to be active in a Gr-dependent manner [59,60]. This activity appeared to correlate with the ability to induce the expression of the cripto-1 gene. It was demonstrated that specific ligand-induced Gr conformational changes induce these effects, with a particular role for the functional groups at the C17 position of the cortisol backbone [60].
Remarkably, an alternative splice variant of Gr was identified in zebrafish, which shows a high level of similarity with the human GR -isoform [57]. In humans, this C-terminal splice variant is not able to bind ligand and has been shown to act as a dominant-negative inhibitor of the canonical GR (called GR ), although this function is debated [61,62]. The zebrafish Gr diverges from Gr at the same point as its human equivalent, and it showed dominantnegative activity in luciferase reporter assays [57,63]. The zebrafish Gr has most likely evolved independently from the human GR , since the amino acid sequence of its isoform-specific Cterminus shows no homology with the human sequence, and the exon encoding this Gr -specific sequence is located at a different position in the gr gene [57]. Further analysis of the role of Gr revealed no dominant-negative activity in vivo, and the function of this splice variant remains unclear [63,64]. and remain stable until at least 7dpf [65]. In-situ hybridization showed no spatial restriction for gr mRNA during embryonic stages, but high expression levels in the brain, liver and intestinal bulb at 5 dpf [57,[66][67][68]. This embryonic expression pattern was confirmed using immunohistochemistry [68]. In adults, expression was demonstrated in spleen, liver, intestine, heart, brain, gill and muscle tissue by RT-PCR [57].
The expression of the zebrafish gr gene has been knocked down using two types of morpholinos: splice-blocking morpholinos that hybridize to a splice site and interfere with splicing at their target site in the genome and translation-blocking morpholinos, which interfere with translation of a transcript by hybridizing to the translation start site. Interestingly, injection of the first type of morpholinos into early zebrafish embryos did not elicit any visible malformations [59,64,67], whereas the latter type resulted in severe developmental defects, like craniofacial and caudal malformations, delayed somitogenesis, and defects in somite and tail morphogenesis [67,68]. These effects were shown to be specific for the gr knockdown, since gr mRNA rescued the phenotypes of the morpholino-treated embryos [67,68]. In addition, morpholino-treated embryos had smaller hearts and reduced cardiac function [69]. The strong effect of the translation-blocking morpholino was explained by its ability to target maternally deposited mRNA, whereas a splice-blocking morpholino only interferes with mRNA which is transcribed de novo, so it is only effective after zygotic transcription has started [67]. Microarray analysis of embryos at 5 and 10 hpf demonstrated that the early gr expression has an important role in cell survival and inhibits apoptosis [67]. At 24 and 36 hpf, a microarray analysis showed disturbed regulation of genes involved in numerous developmental processes in embryos treated with a translation-blocking morpholino [70].
Interestingly, transcriptomic analysis revealed that knockdown of gr expression by a morpholino regulates a different cluster of genes than activation of the receptor by a 6 h treatment with a high dose (100 M) of the synthetic glucocorticoid dexamethasone [64]. These data suggest that Gr regulates two clusters of genes in zebrafish embryos: one that is regulated under basal conditions and one that is regulated upon increased receptor activation, after stress or upon treatment with a pharmacological dose of a glucocorticoid. The first cluster mainly contains genes involved in cell cycle and cell death, whereas the latter cluster mainly contains genes involved in catabolic processes like proteolysis and glycogen breakdown [64]. In support of this hypothesis, in another microarray study treatment with a low dose of dexamethasone (50 nM) between 0 and 5 dpf was found to mainly regulate genes involved in cell cycle and cell death [71].
Finally, ion regulation was severely compromised in embryos upon morpholino knockdown of gr expression. Ca2 + uptake was impaired [72], as well as Na+ uptake following 24 h exposure to acidic water [73]. These results are in line with a decreased number of ionocytes in morpholino-treated embryos, in particular Na + -K + -ATPase rich cells (NaRCs) and H + -ATPase rich cells (HRCs) [74]. Furthermore, the secretion of acid was decreased, which could be explained by a suppressed expression of transporters involved in acid secretion [72].
In a forward-genetic screen of chemically mutated zebrafish a mutation was identified in the gr gene, which results in a single amino acid substitution in the DBD of the receptor, making the Gr transcriptionally inactive [75]. In humans, a mutation in the equivalent amino acid occurs naturally and also results in a transcriptionally inactive receptor and results in resistance to glucocorticoids [76]. This mutant zebrafish shows no morphological malformations, is doi:10.11131/2017/101286 adult-viable and displays increased cortisol levels due to a lack of feedback on the regulation of its secretion. Behavioral studies of adult fish revealed a depression-like profile, including decreased exploratory behavior and impaired habituation to repeated exposure to an anxiogenic environment [75]. Administration of the antidepressant fluoxetine as well as social interactions were shown to restore normal behavior [75,77]. Using this mutant, it was demonstrated that Gr mediates the glucocorticoid-induced inhibition of leukocyte migration upon wounding [78] and the stimulation of the formation of hematopoietic stem and progenitor cells (HSPCs) by glucocorticoids [79]. In addition, it was found that light adaptation in the retina was disturbed in mutant larvae [80]. The relatively mild phenotype of this mutant zebrafish line has cast doubt on the specificity of some of the morphological malformations observed upon morpholino knockdown. This is supported by very recent data from a Crispr/Cas9 gr knockout line, which displays a similarly mild phenotype [81].
Gr activity could be measured using a transgenic line with a luciferase gene driven promoter with four glucocorticoid response elements (GREs). By measuring in-vivo luciferase activity in 5 dpf larvae of this line, the glucocorticoid activity of exogenous compounds could be determined, as well as the action of increased cortisol levels upon exposure to osmotic stress or the administration of the precursor hormone pregnenolone [82]. In two other lines GFP was coupled to a promoter containing six GREs [83] or nine GREs [84]. Ubiquitous fluorescence was detectable from 14 hpf, and at 3 dpf the signal was more specifically expressed in GR target tissues like the brain, liver and pronephros [84]. Due to the high sensitivity Gr activity was also detected in compartments such as fin, eyes, and otic vesicles. In 5 dpf larvae, compounds were screened for glucocorticoid activity, and the specificity of the response was confirmed using morpholino or TALEN knockdown of the receptor or the antagonist RU486 [83,84]. The endogenous cortisol activity was monitored after osmotic stress and during the diurnal cycle [83,84]. In adult fish, fluorescence was observed in many tissues, such as the esophageal sacs mucosa, ventricular epicardium, liver, intestinal mucosa, testis and ovary [84].

The Zebrafish Mr
Finally, in 2011 the zebrafish mr gene was identified, which was shown to be present as a single copy [85]. Using luciferase assays, this receptor was shown to activate the MMTV promoter after binding of aldosterone, cortisol, and 11-deoxycorticosterone (DOC). Fish do not produce aldosterone, but it has been suggested that in fish DOC has a similar function to aldosterone in mammals [86]. DOC is putatively converted from progesterone by steroid 21-monooxygenase (cyp21a1), but the presence of this enzyme has not been demonstrated in zebrafish and has been poorly characterized in several other fish species [43,44]. Interestingly, the human MR antagonist spironolactone showed agonistic activity on the zebrafish Mr, whereas eplerenone and nimodipine acted as antagonists of the zebrafish Mr [85]. A two-hybrid assay showed that aldosterone, cortisol and DOC induced an interaction between the N-and C-terminal ends of the zebrafish Mr, whereas in the human MR this interaction was exclusively induced by aldosterone [85]. Surprisingly, spironolactone did not induce this N/C-interaction, and since this ligand had agonistic activity, the functional significance of the N/C-interaction remains unclear [85].
Little is known about the expression pattern of mr in zebrafish. Expression of mr mRNA was shown by RT-PCR to be minimal immediately after fertilization and increased 52

Future Perspective
An important aspect of steroid receptor research in zebrafish is the relatively limited number of studies that have been performed in this area. A PubMed (https://www.ncbi.nlm.nih. gov/pubmedsearch) search, using 'zebrafish' and the name of the steroid receptors (in as many variations as possible) as search terms, returned a total of 308 published articles, of which more than half involve research on Er (308). For comparison, the same searches with the search term 'mouse' instead of 'zebrafish' retrieved about 50 times more published papers (15,086). This difference is not specific for research on steroid receptors in these animal models and rather reflects the difference in popularity of the models in general, because the terms 'mouse' alone also returned about 50 times more articles than the term 'zebrafish'. Limiting the searches to articles published in 2016 and 2017 showed a 20-fold difference, illustrating an increase in the role of the zebrafish model in steroid receptor research.
In order to further increase the role of the zebrafish model system in steroid receptor research, the advantages of this model system should be better exploited. Morpholino knockdown of the receptor has been performed for all steroid receptor types, but the usage of morpholinos is currently under debate [14]. Fortunately, this technology is rapidly becoming obsolete. Within the Zebrafish Mutation Project (http://www.sanger.ac.uk/resources/zebrafish/zmp/), using a combination of chemical mutagenesis and high-throughput sequencing [28], mutant zebrafish lines have been generated for all steroid receptor types. In addition, targeted mutagenesis has become possible with the CRISPR/Cas9 technology [88], which also allows for the generation of tissue-specific [89] and inducible gene knockout strategies [90]. Another challenge that remains is the analysis of steroid receptor expression at the protein level, due to the limited availability of antibodies specific for (zebra)fish receptors.
Approximately half of the zebrafish steroid receptor research involves ecotoxicological studies on EDCs. The zebrafish will become an increasingly popular model for screening for EDCs due to the identification of novel (molecular) biomarkers and the generation of reporter lines for the activity of steroid receptors (currently, only reporter lines for Er and Gr activity are available). In addition, the increased availability of specific receptor mutants will enhance studies on the mechanism of action of EDCs in the zebrafish model. Furthermore, an increasing role for the zebrafish in biomedical research involving steroid receptors can be expected. Current research aiming at the discovery of novel steroid drugs focuses on the identification of selective receptor modulators, which are steroids that induce desired pharmacological effects without eliciting adverse effects. The zebrafish is highly suitable for in-vivo phenotypic screening and can be used to study compounds that dissociate desired from adverse effects. Targeted mutagenesis of receptor domains involved in e.g. DNA binding, dimerization or interaction with transcriptional coregulators will further advance our understanding of the specific molecular actions of steroid drugs, and how they translate into their desired and adverse pharmacological effects. In these ecotoxicological and biomedical studies the evolutionary distance of the zebrafish from humans doi:10.11131/2017/101286 compared to mammalian model organisms like mice and rats should be taken into account. Data obtained in zebrafish will often need validation in more closely related animal models before they can be extrapolated to humans. Therefore, the zebrafish model should not be considered as a model organism replacing other animal models, but rather as an additional complementary tool with specific advantages.
In conclusion, the initial characterization and expression analysis of all five steroid receptor types, the generation of mutants and reporter lines and the identification of biomarkers to study their action has made the zebrafish an ideal model system for research on steroid receptors. It is therefore anticipated that in the next decade the role of the zebrafish in steroid receptor research will significantly increase, particularly in research on EDCs and selective steroid receptor modulators.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
89Zr-Cobalamin PET Tracer: Synthesis, Cellular Uptake, and Use for Tumor Imaging

Vitamin B12, or cobalamin (Cbl), is an essential nutrient. Acquisition, transport, and cellular internalization of Cbl are dependent on specific binding proteins and associated receptors. The circulating transport protein transcobalamin (TC) promotes cellular uptake via binding to specific receptors such as CD320, a receptor upregulated in several cancer cell lines. In this study, we report the successful synthesis of 89Zirconium-labeled Cbl that was derivatized with desferrioxamine (89Zr-Cbl). We document the purity of the tracer and its binding to TC compared with that of unmodified cyano-Cbl (CN-Cbl). In vitro studies employing the CD320 receptor-positive breast cancer cell line MDA-MB-453 showed a 6- to 10-fold greater uptake of 89Zr-Cbl when compared with the uptake in the presence of 200-fold excess of CN-Cbl at 37 °C. We used nude mice with MDA-MB-453 tumors to study the feasibility of employing the tracer to visualize CD320 positive tumors. In vivo positron emission tomography images displayed a clear visualization of the tumor with 1.42 ± 0.48 %ID/g uptake (n = 3) at 4 h after injection (p.i.) with the tracer retained at 48 h p.i. Ex vivo biodistribution studies using 89Zr-Cbl exhibited the highest uptake in kidney and liver at 48 h p.i. Results document the feasibility of synthesizing a Cbl-based tracer suitable for both in vivo and ex vivo studies of Cbl trafficking and with the potential to visualize tumors expressing TC receptors, such as CD320.

■ INTRODUCTION
Vitamin B 12 (cobalamin, Cbl) is a critical nutrient that is physiologically required to maintain cell growth and differentiation. 1−4 Cbl is involved in the biosynthesis of nucleic acids, lipids, and proteins, and its deficiency leads to a reduction in functional methionine synthase and metabolism of methylmalonic acid in humans, leading to megaloblastic anemia and/ or various neurological disorders. 1−4 Cbl gains entry into cells upon binding to transport proteins and subsequent receptor mediated transport. Cbl in blood is bound to the transport protein transcobalamin (TC) (holo-TC), which, in turn, is recognized by specific receptors such as CD320. 1−4 Upregulation of CD320 receptors has been reported in several malignancies including breast, prostate, thyroid, cervical, colorectal, and stomach cancers. 5 The important role of Cbl in cellular proliferation and the upregulation of CD320 in tumor cells has made Cbl uptake an attractive candidate for tumor imaging, mainly using singlephoton emission computed tomography with 99m Technetiumor III Indium-labeled Cbl. 6−13 One Cbl-positron emission tomography (PET) imaging agent, labeled with 64 Cu (t 1/2 ∼ 12.7 h), has also been reported. 14 Herein, the utility of Cbl as a vector was explored for delivering the PET radionuclide 89 Zr (t 1/2 ∼ 3.27 days). We hypothesized that 89 Zr would (1) retain the sensitivity of PET imaging and (2) provide a longer visualization window by providing a greater signal-to-noise ratio compared with prior tracers reported, allowing for an improved tumor targeting and imaging. Following radiosynthesis, we evaluated the in vitro uptake and in vivo pharmacokinetics of the CD320-positive MDA-MB-453 breast cancer in athymic nude mice.

■ RESULTS
Synthesis of Cbl-Desferrioxamine (Cbl-DFO). Cbldesferrioxamine (Cbl-DFO) was synthesized by forming a carbamate linkage between the 5′-hydroxyl of deoxyribose moiety in Cbl and the amine group of DFO (Scheme 1, Figure  S1). 15 Cbl was activated using 1,1′-carbonyl-di-(1,2,4-triazole) (CDT), followed by the addition of DFO, which links through the primary amine. Purification and characterization confirmed that the conjugate was of ≥97% purity ( Figures S2−S4 Radiolabeling of Cbl with 89 Zr. Cbl-DFO was labeled with 89 Zr ( 89 Zr-Cbl) using a previously established protocol (Scheme 1). 16 A radiolabeling efficiency of ∼97% was determined by instant thin-layer chromatography (iTLC, Figure S5a). The specific activity of the tracer was determined by titrating 89 Zr 4+ and Cbl at different mole ratios with an
In Vitro Stability of 89 Zr-Cbl. Stability of the tracer was analyzed by incubating 89 Zr-Cbl in saline and in human serum at 37°C. Bound versus unbound radio metal was analyzed at 0, 4, 24, and 48 h after incubation using iTLC (Figure 1a,b). The intact tracer was located closer to the origin (40−80 mm), whereas unbound tracer was found at 100−140 mm. After 48 h of incubation, free 89 Zr was <1% in both saline and serum.
TC Binding Studies. Mouse TC binding of 91 Zr-Cbl was studied by radiometric chase assay using 57 Co-labeled Cbl employing a previously described design. 17 91 Zr-Cbl was synthesized similar to 89 Zr-Cbl, but with 91 ZrCl 4 . Mouse TC binding of 91 Zr-Cbl displaced 57 Co-labelled Cbl in a manner comparable to that of CN-Cbl (Figure 1c), indicating that the modification of Zr-Cbl did not compromise binding to TC. The same results were obtained for binding to human intrinsic factor (data not shown).

ACS Omega
Article versus 83 ± 15 cpm/10 5 cells at 24 h (p < 0.0001). Competitive assays using excess Cbl at 37°C displayed lower binding at all time points (p < 0.01). All of the data are reported as mean ± standard deviation of four independent measurements.

■ DISCUSSION
In this proof-of-concept study, we report the successful production of a Cbl-derived 89 Zr tracer suitable for use in PET studies. Studies on nude mice bearing a human breast cancer cell tumor allow us to demonstrate the use of the tracer for PET visualization of the tumor.
In agreement with previous data, 17,18 we found that 91 Zr-Cbl bound to mouse TC in a manner comparable to that of CN-Cbl ( Figure 1c). Next, an in vitro assay was performed in the breast cancer cell line MDA-MB-453 to demonstrate a specific uptake of 89 Zr-Cbl. 19 We demonstrated a greater than 4-fold uptake of 89 Zr-Cbl at 37°C versus 4°C; blocking with 200-fold excess unlabeled Cbl had a similar reduced uptake (Figure 1d). These results support the idea that the targeting properties of 89 Zr-Cbl in MDA-MB-453 cells rely on a Cbl-dependent internalization mechanism, likely through the CD320 receptor.
In vivo imaging with 89 Zr-Cbl showed an uptake in MDA-MB-453 tumors with 1.42 ± 0.48 of %ID/g, whereas ex vivo tissue distribution studies showed a tumor uptake of 5.11 ± 1.33 %ID/g at 4 h p.i. (Figures 2 and 3). Notable uptake was also observed in the liver and kidneys with 4.27 ± 0.51 and 8.92 ± 1.45 %ID/g at 4 h p.i., respectively (Figure 2c). An in vivo block using 200-fold excess of unradiolabeled Cbl showed a significantly reduced uptake (p < 0.001) of the tracer, indicating that the in vivo tumor initialization is Cbl dependent, supported in the in vitro internalization assay. Muscle-to-tumor ratio showed that the maximum tumor-to-background ratio was achieved at 4 h p.i.
To compare the uptake values with those described in the literature, the biodistribution data will be used for a more accurate comparative analysis for the rest of the discussion. Tumor uptake persisted throughout the 4−48 h imaging period without a significant change (5.11 ± 1.33 at 4 h vs 3.78 ± 0.77 at 48 h, p > 0.1), whereas blood clearance was evident between 4 and 24 h with approximately ∼80% decrease (5.28 ± 0.62 at 4 h vs 0.92 ± 0.25 %ID/g at 24 h) in the circulating 89 Zr-Cbl; the final blood activity was observed to be 0.39 ± 0.06 %ID/g at 48 h p.i.
The tumor uptake achieved in our model is comparable to that of the other Cbl-based tracers reported thus far. 6−14 Ikotun et al. investigated the tumor uptake of 64 Cu-labeled Cbl in pancreatic, ovarian, murine melanoma, and colorectal tumor models, with the highest %ID/g being 4.84 ± 0.32 at 6 h p.i. in the colorectal tumor models. 14 In the melanoma model, the tumor uptake was highest (3.43 ± 0.87 %ID/g) at 1 h, which decreased to 2.64 ± 0.10 %ID/g after 24 h, whereas 89 Zr-Cbl had a higher accumulation and did not show a significant decrease in tumor accumulation over 4−48 h, p > 0.1.
One of the limitations of our 89 Zr-Cbl tracer is the observed kidney uptake (94.42 ± 4.27 %ID/g at 4 h), a problem across all of the Cbl tracers to date. Renal processing is the most prominent route for Cbl accumulation/storage and/or reabsorption and is driven by megalin, a known TC-Cbl receptor expressed in kidney proximal tubuli. 20,21 In addition, this is the first positively charged PET tracer reported, the effect of which is unknown. An overall positive charge on the Cbl (as 89 Zr-Cbl conjugate), or indeed simply the result of modification of the β-axial position, may affect the Cbl cellular processing across tissues, as has been shown previously for two forms with varying α-ligands (OH-Cbl and CN-Cbl) Cbl species, and warrants further investigation. 12,22−24 89 Zr-Cbl shows feasibility as a PET tracer to identify MDA-MB-453 tumors in vivo. The longer window for PET imaging allowed for reduced uptake in the kidneys, a problem to date in Cbl radiotracers, while still maintaining a moderate tumor uptake over 48 h p.i. This tracer is promising since, to our knowledge, tumor uptake is the highest reported to date for a B12 based PET probe.

■ CONCLUSIONS
We have successfully developed and evaluated the first 89 Zrlabeled Cbl tracer as a viable tool for visualizing TC-mediated Cbl uptake into a CD320 positive tumor. 89 Zr-Cbl displayed retained tumor uptake up to 48 h p.i., allowing for a longer imaging window. Our data paves the road for future studies to understand the kinetics of Cbl transport and to study the use as a tool for visualizing tumors capable of accumulating Cbl. 89 Zr-Radiochemistry. Optimum conditions for radiolabeling of Cbl-DFO were tested by titrating with 89 Zr and analyzing the incubated solution using iTLC. Optimum labeling activity was found to be 250 ± 20 mCi/μmol (9250 ± 740 MBq/ micromole). Approximately 1 mCi (37 MBq) of 89 Zr(C 2 O 4 ) 2 (3D Imaging, LLC) was diluted with 0.9% saline and the pH was adjusted to 7−7.5 by adding 1 M Na 2 CO 3 . A solution of Cbl-DFO (0.004 μmol, 10.8 μg) was added to the pH-adjusted 89 Zr solution and incubated for 20 min at ambient temperature (Scheme 1). Radiolabeling efficiency of >97% was determined by iTLC using silica iTLC strips and EDTA mobile phase ( Figure S5a). The identity of the tracer was characterized via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis using Cbl-DFO labeled with cold Zr 4+ as standard. Expected: 2030.  Figure S5b).
Mouse TC Binding to 91 Zr-Cbl. Nonradioactive 91 Zr-Cbl was synthesized for TC binding studies by reacting Cbl-DFO with 91 ZrCl 4 as described above. The conjugate was characterized by ESI-MS (data not shown). Mouse TC binding of 91 Zr-Cbl was confirmed by radiometric chase assay using 57 Co-labeled Cbl and compared with free Cbl (cyanocobalamin) employing a previously described protocol. 17 Mouse TC was derived as previously described. 25 In Vitro Uptake. Modified internalization assay was performed. MDA-MB-453 cells were cultured in Cbl-free media (DMEM with 10% charcoal stripped FBS) and plated in six-well plates. Each well contained 200 000 cells plated and incubated overnight. To each well, 89 Zr-Cbl (0.1 μCi, 3.7 KBq, 0.4 pmol of Cbl per well) was added. For the blocking experiment, unmodified Cbl was added (40 pmol per well). Plates were incubated for 1, 4, and 24 h intervals at either 37 or 4°C. At the end of each time point, wells were serially washed with phosphate-buffered saline (1×), acid (1 mM acetic acid and 1 mM glycine), and base (1 M NaOH, 1 mL, 5 min). Each wash was collected and measured for bound activity using a γ counter (Perking Elmer 2480 WIZARD). Control wells were trypsinized and counted using a cell counter (Contessa II). Internalized activity was normalized to 10 5 cells.
Cell Lines and Small Animal Xenografts. All of the animal handling and manipulations were conducted in accordance with the guidelines set by WSU Animal Care and Use Committee (IACUC). For imaging and in vivo uptake experiments, female nude mice (Envigo) were kept under Cbldeficient diet (Teklad Cbl-free custom diet, Envigo) for 3 weeks. Cells were subcutaneously implanted on the shoulder with MDA-MB-453 cancer cells (5 × 10 6 cells/mouse) after 2 weeks of Cbl-free diet. Cells were injected in 1:1 media/ matrigel (Corning LLC) at a volume of 200 μL. The tumor volume until was calculated using the formula length × width 2 × 0.52. Mice with tumors of 100−200 mm 3 dimensions were used for imaging experiments.
Ex Vivo Distribution and Competitive Saturation. The tissue distribution of 89 Zr-Cbl was studied by administering 10−25 μCi (0.37−0.93 MBq, 0.04−0.1 nmol) of the tracer on the lateral tail vain of the rodent. For the competitive saturation assay, ∼20 nmol/mouse of cold CN-Cbl was co-injected with 89 Zr-Cbl. Euthanasia was performed via CO 2 asphyxiation at 4, 24, and 48 h p.i.


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The Application of a Modified d-ROMs Test for Measurement of Oxidative Stress and Oxidized High-Density Lipoprotein

Reactive oxygen species (ROS) are involved in the initiation and progression of atherosclerosis. ROS-derived hydroperoxides, as an indicator of ROS production, have been measured by using the diacron reactive oxygen metabolites (d-ROMs) test, which requires iron-containing transferrin in the reaction mixture. In this study we developed a modified d-ROMs test, termed the Fe-ROMs test, where iron ions were exogenously added to the reaction mixture. This modification is expected to exclude the assay variation that comes from different blood iron levels in individuals. In addition, this Fe-ROMs test was helpful for determining the class of plasma lipoproteins that are hydroperoxidized. Low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and high-density lipoprotein (HDL) were purified by use of an LDL/VLDL purification kit and the dextran sulfate-Mg2+ precipitation method, respectively; their hydroperoxide contents were assessed by performing the Fe-ROMs test. The majority of the hydroperoxides were detected only in the HDL fraction, not in the LDL/VLDL. Further detailed analysis of HDLs by size-exclusion high-performance liquid chromatography revealed that the hydroperoxide-containing molecules were small-sized HDLs. Because HDL was shown to be the principal vehicle for the plasma hydroperoxides, this Fe-ROMs test is a beneficial method for the assessment of oxidized-HDL levels. Indeed, Fe-ROMs levels were strongly associated with the levels of oxidized HDL, which were determined by performing the malondialdehyde-modified HDL enzyme immunoassay. In conclusion, the Fe-ROMs test using plasma itself or the HDL fraction after dextran sulfate-Mg2+ precipitation is useful to assess the functionality of HDL, because the oxidation of HDL impairs its antiatherogenic capacity.

Introduction
Oxidative stress can be caused by excess reactive oxygen species (ROS) and reactive nitrogen species. ROS include superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( • OH), and singlet oxygen ( 1 O 2 ). They are generated by the consumption of O 2 and thought to cause deleterious oxidative damage to virtually all molecules. These reactive species are not necessarily a threat to the body under normal physiological conditions [1,2], but if the body is not able to remove them to a . The results are expressed as the mean ± standard deviation (n = 5). A symbol (*) represents a statistically significant difference (p < 0.001). When the plasma samples from two other healthy subjects were used, similar effects of Fe ions were observed.

The Strong Correlation between Levels of Hydroperoxides Measured by the d-ROMs and Fe-ROMs Tests
We next measured the levels of hydroperoxide in the plasma from 10 subjects with experience of acute febrile disease by using the original d-ROMs test and our modified one (the Fe-ROMs test). For this experiment, we chose subjects with acute febrile disease because they show a wide range of d-ROMs levels from normal to high. Indeed, their hydroperoxides levels ranged from 205 to 430 Carratelli units (U.CARR) ( Figure 2). The levels obtained by the original and the Fe-ROMS tests showed a strong correlation (r = 0.986), although the ratios of Fe-ROMs levels to d-ROMs levels tended to become higher as the d-ROMs levels increased. The correlation between both levels was also strong in five healthy subjects (r = 0.896, Figure S1). of absorbance changes at 505 nm after the addition of plasma from a healthy subject and N,N -diethylp-phenylenediamine were determined in the presence ( ) and absence ( ) of 100 µM Fe 2+ as described in the (Section 4); (b) Increased rate of absorbance (mOD/min) during 120 to 300 s was determined after the addition of N,N -diethyl-p-phenylenediamine, and iron ions (Fe 2+ or Fe 3+ ) in the presence of plasma (Experiment 1) or in the presence or absence of plasma (Experiment 2). The results are expressed as the mean ± standard deviation (n = 5). A symbol (*) represents a statistically significant difference (p < 0.001). When the plasma samples from two other healthy subjects were used, similar effects of Fe ions were observed.

The Strong Correlation between Levels of Hydroperoxides Measured by the d-ROMs and Fe-ROMs Tests
We next measured the levels of hydroperoxide in the plasma from 10 subjects with experience of acute febrile disease by using the original d-ROMs test and our modified one (the Fe-ROMs test). For this experiment, we chose subjects with acute febrile disease because they show a wide range of d-ROMs levels from normal to high. Indeed, their hydroperoxides levels ranged from 205 to 430 Carratelli units (U.CARR) ( Figure 2). The levels obtained by the original and the Fe-ROMS tests showed a strong correlation (r = 0.986), although the ratios of Fe-ROMs levels to d-ROMs levels tended to become higher as the d-ROMs levels increased. The correlation between both levels was also strong in five healthy subjects (r = 0.896, Figure S1).

The Fe-ROMs Test Detects the Oxidation of Plasma Molecules of High Molecular Weight
The Fe-ROMs test has some advantages over the original one. In the original test, both hydroperoxides and iron ion are supplied by plasma; but in the Fe-ROMs test, iron ions are supplied exogenously in the reaction mixture. Therefore, the Fe-ROMs test allowed us to fractionate hydroperoxide-containing molecules from plasma without paying attention to the presence of ironsupplying molecules. We first separated plasma into filtrate and residual fluid by using an Amiconultra-0.5 (100 kDa). The recovery of hydroperoxides in the residual fluid, which was determined by the Fe-ROMs test, was 99.3% (the average recovery of three independent experiments), indicating that most of the hydroperoxidized molecules were included among the macromolecular components.

Defining the Macromolecular Components Oxidized in Plasma: Oxidized Macromolecular Components Are Not Low-Density Lipoprotein/Very Low-Density Lipoprotein (LDL/VLDL)
Oxidative stress targets a variety of molecules in plasma. Among them, lipids, especially PUFAs, are most subjected to oxidative stress, leading to lipid peroxidation. Lipoproteins are macromolecular complexes of phospholipids, free cholesterol, cholesterol esters, triglycerides, and apolipoproteins. Because phospholipids, cholesterol esters, and triglycerides contain PUFAs, it is most likely that hydroperoxides are present in plasma lipoproteins. We then isolated the low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) fraction from the plasma by using an LDL/VLDL Purification Kit. A dextran solution was first added to the plasma to selectively precipitate LDL/VLDL from the plasma, and the pellet obtained by centrifugation was used for the subsequent LDL/VLDL purification. We detected less than 3% of hydroperoxides in the purified LDL/VLDL as compared to the amount in the original plasma sample, indicating that the hydroperoxide level in LDL/VLDL was too low to be detected by the Fe-ROMs test. In contrast, all the activity (105%) was detected in the supernatant obtained after centrifugation following the addition of the Dextran solution, indicating that HDL was probably the major carrier of hydroperoxides in plasma.

The Oxidation of HDL Is Detected by the Fe-ROMs Test
We then isolated the HDL from the other lipoproteins with a combination of dextran sulfate and Mg 2+ , and assayed its activity by performing the Fe-ROMs test. All the activity (104%) was recovered into the supernatant after the dextran sulfate-Mg 2+ precipitation procedure, suggesting that the hydroperoxide-containing molecule was HDL. The lipoprotein separation was quite sensitive to the The oxidative stress was evaluated by measuring plasma samples from 10 male patients. The rates of increase in absorbance at 505 nm were determined in the presence (Fe-ROMs test) and absence (d-ROMs test) of Fe 2+ , as described in the Section 4. The correlation between the values obtained from the d-ROMs and Fe-ROMs tests was expressed by Pearson's correlation coefficient (r = 0.986, p < 0.001). U.CARR, Carratelli units.

The Fe-ROMs Test Detects the Oxidation of Plasma Molecules of High Molecular Weight
The Fe-ROMs test has some advantages over the original one. In the original test, both hydroperoxides and iron ion are supplied by plasma; but in the Fe-ROMs test, iron ions are supplied exogenously in the reaction mixture. Therefore, the Fe-ROMs test allowed us to fractionate hydroperoxide-containing molecules from plasma without paying attention to the presence of iron-supplying molecules. We first separated plasma into filtrate and residual fluid by using an Amiconultra-0.5 (100 kDa). The recovery of hydroperoxides in the residual fluid, which was determined by the Fe-ROMs test, was 99.3% (the average recovery of three independent experiments), indicating that most of the hydroperoxidized molecules were included among the macromolecular components. Oxidative stress targets a variety of molecules in plasma. Among them, lipids, especially PUFAs, are most subjected to oxidative stress, leading to lipid peroxidation. Lipoproteins are macromolecular complexes of phospholipids, free cholesterol, cholesterol esters, triglycerides, and apolipoproteins. Because phospholipids, cholesterol esters, and triglycerides contain PUFAs, it is most likely that hydroperoxides are present in plasma lipoproteins. We then isolated the low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) fraction from the plasma by using an LDL/VLDL Purification Kit. A dextran solution was first added to the plasma to selectively precipitate LDL/VLDL from the plasma, and the pellet obtained by centrifugation was used for the subsequent LDL/VLDL purification. We detected less than 3% of hydroperoxides in the purified LDL/VLDL as compared to the amount in the original plasma sample, indicating that the hydroperoxide level in LDL/VLDL was too low to be detected by the Fe-ROMs test. In contrast, all the activity (105%) was detected in the supernatant obtained after centrifugation following the addition of the Dextran solution, indicating that HDL was probably the major carrier of hydroperoxides in plasma.

The Oxidation of HDL Is Detected by the Fe-ROMs Test
We then isolated the HDL from the other lipoproteins with a combination of dextran sulfate and Mg 2+ , and assayed its activity by performing the Fe-ROMs test. All the activity (104%) was recovered into the supernatant after the dextran sulfate-Mg 2+ precipitation procedure, suggesting that the hydroperoxide-containing molecule was HDL. The lipoprotein separation was quite sensitive to the molecular size of the dextran sulfate, the concentrations of dextran sulfates and Mg 2+ , and the temperature. Consequently, we evaluated the lipoprotein profiles of the plasma and the supernatant after performing the dextran sulfate-Mg 2+ precipitation by using gel-permeation high-performance liquid chromatography (GP-HPLC). As shown in Figure 3 and Table 1, the lipoprotein profiles revealed that the plasma contained four major classes of lipoproteins including VLDL, LDL, and HDL, but the supernatant contained only HDL. More detailed analysis of the lipoproteins (Table 1), in which each of these four major classes were divided into sub-classes on the basis of the lipoprotein particle size, shows that all sizes of HDL were recovered in the supernatant. Furthermore, the supernatant contained only small amounts of other lipoproteins including small LDL and very small LDL. This is an important finding, because a small dense LDL, which is associated with an increased risk of atherosclerotic disease, has been reported to be vulnerable to oxidation [28][29][30]. after performing the dextran sulfate-Mg 2+ precipitation by using gel-permeation high-performance liquid chromatography (GP-HPLC). As shown in Figure 3 and Table 1, the lipoprotein profiles revealed that the plasma contained four major classes of lipoproteins including VLDL, LDL, and HDL, but the supernatant contained only HDL. More detailed analysis of the lipoproteins (Table 1), in which each of these four major classes were divided into sub-classes on the basis of the lipoprotein particle size, shows that all sizes of HDL were recovered in the supernatant. Furthermore, the supernatant contained only small amounts of other lipoproteins including small LDL and very small LDL. This is an important finding, because a small dense LDL, which is associated with an increased risk of atherosclerotic disease, has been reported to be vulnerable to oxidation [28][29][30]. The HDL fraction was isolated from the plasma of a healthy subject by using the dextran sulfate/Mg 2+ method as described in the Section 4. The plasma and isolated HDL fractions were analyzed by using a dual detection GP-HPLC system for lipoprotein analysis as described in the Section 4. Cholesterol and triglycerides concentrations were monitored for lipoprotein profiling. The retention times of the low-density lipoprotein (LDL) and HDL standard materials were 23.2 and 26.0 min, respectively.  The HDL fraction was isolated from the plasma of a healthy subject by using the dextran sulfate/Mg 2+ method as described in the Section 4. The plasma and isolated HDL fractions were analyzed by using a dual detection GP-HPLC system for lipoprotein analysis as described in the Section 4. Cholesterol and triglycerides concentrations were monitored for lipoprotein profiling. The retention times of the low-density lipoprotein (LDL) and HDL standard materials were 23.2 and 26.0 min, respectively.

The Fe-ROMs Test Is a Useful Method for the Determination of Oxidized High-Density Lipoprotein (HDL)
As described above, almost all the plasma hydroperoxides were detected in the HDL fraction. Because the levels of Fe-ROMs in the plasma were different from subject to subject, it was interesting for us to compare Fe-ROMs levels in the plasma and its dextran sulfate-supernatant in many subjects. Figure 4 shows that the correlation between Fe-ROMs levels in both samples from six healthy men was very strong. Further, a significant correlation was maintained between both samples from 10 subjects with acute febrile disease (r = 0.924, Figure S2), indicating that the hydroperoxidized HDL in many subjects can be estimated from the plasma's Fe-ROMs levels. As described above, almost all the plasma hydroperoxides were detected in the HDL fraction. Because the levels of Fe-ROMs in the plasma were different from subject to subject, it was interesting for us to compare Fe-ROMs levels in the plasma and its dextran sulfate-supernatant in many subjects. Figure 4 shows that the correlation between Fe-ROMs levels in both samples from six healthy men was very strong. Further, a significant correlation was maintained between both samples from 10 subjects with acute febrile disease (r = 0.924, Figure S2), indicating that the hydroperoxidized HDL in many subjects can be estimated from the plasma's Fe-ROMs levels. Figure 4. The correlation between the Fe-ROMs levels in the plasma and HDL fraction. HDL fractions were isolated from the plasma of six healthy subjects (aged 27-69 years), as described in Figure 3. The Fe-ROMs values were then measured by using these plasma samples and isolated HDL fractions. The correlation between the Fe-ROMs levels in the plasma and isolated HDL fractions is expressed by Pearson's correlation coefficient (r = 0.981, p = 0.001).

Small-Sized HDL Is a Major Oxidized Molecule in Plasma
We next fractionated the plasma from a healthy subject by using HPLC and defined the molecules that had been hydroperoxidized. As shown in Figure 5a,b, the highest peak (eluted at 17.065 min and collected into Fraction 18) and the second highest one (16.234 min and Fraction 17) were identified as albumin and IgG, respectively, by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) analysis. On the other hand, HDLs were eluted in Fractions 14-17 ( Figure 5c). However, hydroperoxide-containing molecules were not eluted coincidentally with the majority of HDLs and were detected only in Fraction 17 (Figure 5d). Because not only small HDL but also IgG were included in Fraction 17, we examined the involvement of IgG in the activity. As summarized in Figure 6, when IgG was removed from the plasma by protein G column chromatography, the recovery of the Fe-ROMs level in IgG-depleted plasma was 80.1%. Because we often experienced a loss of activity during the concentration procedure using Amicon Ultra centrifugation, we concluded that IgG had no critical effect on the Fe-ROMs levels. Therefore, we considered HDL, in particular the small HDL, most likely to be the major carrier of hydroperoxides in plasma. The correlation between the Fe-ROMs levels in the plasma and HDL fraction. HDL fractions were isolated from the plasma of six healthy subjects (aged 27-69 years), as described in Figure 3. The Fe-ROMs values were then measured by using these plasma samples and isolated HDL fractions. The correlation between the Fe-ROMs levels in the plasma and isolated HDL fractions is expressed by Pearson's correlation coefficient (r = 0.981, p = 0.001).

Small-Sized HDL Is a Major Oxidized Molecule in Plasma
We next fractionated the plasma from a healthy subject by using HPLC and defined the molecules that had been hydroperoxidized. As shown in Figure 5a,b, the highest peak (eluted at 17.065 min and collected into Fraction 18) and the second highest one (16.234 min and Fraction 17) were identified as albumin and IgG, respectively, by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) analysis. On the other hand, HDLs were eluted in Fractions 14-17 ( Figure 5c). However, hydroperoxide-containing molecules were not eluted coincidentally with the majority of HDLs and were detected only in Fraction 17 (Figure 5d). Because not only small HDL but also IgG were included in Fraction 17, we examined the involvement of IgG in the activity. As summarized in Figure 6, when IgG was removed from the plasma by protein G column chromatography, the recovery of the Fe-ROMs level in IgG-depleted plasma was 80.1%. Because we often experienced a loss of activity during the concentration procedure using Amicon Ultra centrifugation, we concluded that IgG had no critical effect on the Fe-ROMs levels. Therefore, we considered HDL, in particular the small HDL, most likely to be the major carrier of hydroperoxides in plasma.

The Oxidation of HDL Can Be Measured by the Malondialdehyde (MDA)-HDL Enzyme-Linked Immunosorbent Assay and the Fe-ROMs Test
The oxidation of HDL causes a reduction in the ability of HDL to promote cholesterol efflux [21][22][23]. Therefore, monitoring circulatory levels of oxHDL is critical to the diagnosis of atherosclerosis. Oxidative stress generates hydroperoxides in HDL and subsequently forms reactive aldehyde products, such as MDA. MDA attaches covalently to the amino groups of lysine residues of HDL protein components (MDA-HDL). Therefore, an enzyme-linked immunosorbent assay (ELISA) for MDA-HDL has been developed for the determination of oxHDL. We obtained HDL fractions from six healthy male subjects by using the dextran-sulfate-Mg 2+ precipitation procedure and determined their oxidation levels by performing an MDA-HDL enzyme immunoassay and Fe-ROMs test (Figure 7). Both levels were strongly correlated (r = 0.864), although two subjects with similar Fe-ROMs levels (about 20 mOD/min) showed different MDA-HDL levels.

The Oxidation of HDL Can Be Measured by the Malondialdehyde (MDA)-HDL Enzyme-Linked Immunosorbent Assay and the Fe-ROMs Test
The oxidation of HDL causes a reduction in the ability of HDL to promote cholesterol efflux [21][22][23]. Therefore, monitoring circulatory levels of oxHDL is critical to the diagnosis of atherosclerosis. Oxidative stress generates hydroperoxides in HDL and subsequently forms reactive aldehyde products, such as MDA. MDA attaches covalently to the amino groups of lysine residues of HDL protein components (MDA-HDL). Therefore, an enzyme-linked immunosorbent assay (ELISA) for MDA-HDL has been developed for the determination of oxHDL. We obtained HDL fractions from six healthy male subjects by using the dextran-sulfate-Mg 2+ precipitation procedure and determined their oxidation levels by performing an MDA-HDL enzyme immunoassay and Fe-ROMs test ( Figure  7). Both levels were strongly correlated (r = 0.864), although two subjects with similar Fe-ROMs levels (about 20 mOD/min) showed different MDA-HDL levels. Figure 7. The measurement of oxidized HDL levels by the Fe-ROMs test and the enzyme-linked immunosorbent assay (ELISA). The HDL fractions were isolated from the plasma of six healthy subjects (aged 27-69 years) by using the dextran sulfate-Mg 2+ method as described in the Section 4. Each of the isolated HDL fractions was used for the measurement of HDL peroxides by use of the Fe-ROMs test and for the measurement of MDA-HDL by use of the ELISA. The correlation between the levels of oxidized HDL, which were measured by the Fe-ROMs test and the ELISA, is expressed by Pearson's correlation coefficient(r = 0.864, p = 0.027).

Plasma HDL Concentrations in Subjects with Different HDL Oxidation Levels
As shown in Figure 7, the oxHDL levels differed among the six subjects examined. Because the difference may have occurred due to different plasma HDL concentrations in them, we next determined the concentrations of HDL in four subjects, two of which had higher levels of oxHDL, and the others had lower ones. The lipoprotein profiles (Table 2) and more detailed analysis of HDL (Table 3) indicated no significant difference in the HDL concentrations between four subjects. Thus, the subjects with higher Fe-ROMs values had more hydroperoxidized lipids per HDL molecule.

Plasma HDL Concentrations in Subjects with Different HDL Oxidation Levels
As shown in Figure 7, the oxHDL levels differed among the six subjects examined. Because the difference may have occurred due to different plasma HDL concentrations in them, we next determined the concentrations of HDL in four subjects, two of which had higher levels of oxHDL, and the others had lower ones. The lipoprotein profiles (Table 2) and more detailed analysis of HDL (Table 3) indicated no significant difference in the HDL concentrations between four subjects. Thus, the subjects with higher Fe-ROMs values had more hydroperoxidized lipids per HDL molecule.

Discussion
Because there is a close association between oxidative stress and vascular diseases, we need reliable biomarkers to assess such stress. In the present study we assessed oxidative stress in human plasma by use of a modified d-ROMs test (i.e., the Fe-ROMs test) where iron ions were exogenously added to the reaction mixture. Our Fe-ROMs test had some advantages over the original d-ROMs test: (1) The Fe-ROMs test required a smaller volume of plasma to measure oxidative stress; (2) it gave us an opportunity to define hydroperoxidized target molecules in the blood; and (3) it could exclude the assay variation that comes from different blood iron levels in individuals.
The d-ROMs test is a simple assay marketed for analyzing the total amount of hydroperoxides in serum via Fenton's reaction. However, earlier reports have indicated that the signal detected in this assay is affected by blood components such as iron ions [31]. The serum iron reference range is about 55-160 µg/dL in men and 40-155 µg/dL in women. Therefore, if we use plasma from people with normal serum iron values (40-160 µg/dL) for the determination of d-ROMs levels, iron in the reaction mixture is calculated at a concentration from 0.14 to 0.57 µM. We have observed that d-ROMs levels are increased by iron ions in a dose-dependent manner up to 100 µM ( Figure S1). Thus, d-ROMs levels depend on the levels of hydroperoxides and iron ions in plasma. We anticipated that Fe-ROMs levels are not necessarily correlated with those of d-ROMs. However, the correlation between the levels obtained by the d-ROMs and Fe-ROMS tests was strong in subjects with acute febrile disease, although it tended to be non-linear. This non-linearity may indicate that exogenous iron ions are required for the measurement of blood samples containing high levels of hydroperoxides. In the d-ROMs test, iron ions are supplied mostly from Fe 3+ -binding transferrin protein in blood samples. The affinity of transferrin for ferric iron (Fe 3+ ) is extremely high under the extracellular environment (approximately pH 7.4), and weakened in the acidic conditions, leading to Fe 3+ dissociation from the protein [32]. It thus appears that Fe 3+ rather than Fe 2+ is mainly used in the acidic conditions of the d-ROMs test. Fe 3+ increased absorbance at 505 nm dose-dependently, but to a lesser extent than Fe 2+ . Therefore, the d-ROMs test might not be strongly influenced by different blood iron levels in individuals. However, we recommend adding exogenous iron ions to the reaction mixture, because adding even a small amount of Fe 3+ (i.e., 1 µM) to the reaction mixture changed the absorbance at 505 nm ( Figure S3).
There are some criticisms about the reliability of the d-ROMs test as a method for estimating oxidative stress in a serum or plasma, as the concentration of some ions (i.e., Fe, Cu, etc.) in blood samples might interact in the assay reaction and influence the results [31]. Recent studies have excluded the critical interference of these ions on the d-ROMs test [33,34]; the correlation between the concentration of iron ions and the d-ROMs readings was non-significant in 502 bovine samples. However, in the Fe-ROMs test, not only iron ion in blood samples but also exogenous iron ion was present in the reaction mixture. Hydroperoxides in the reaction mixture undergo iron-mediated one-electron reduction and form free radicals, which react with N,N -diethyl-p-phenylenediamine to form a colored radical that is detectable at 505 nm. Concomitantly, the formation of free radicals can initiate a radical chain reaction with the hydrocarbon part of an unsaturated lipid molecule. In this study we have observed a continuous increase in absorbance at 505 nm at least until 10 min irrespective of the presence or absence of iron ions (Figure 1). This observation may indicate that the absorbances in the d-ROMs and Fe-ROMs tests were increased in a similar fashion via a radical chain reaction, although their increased rates were very different from each other.
ROS oxidize not only lipids but also other biological molecules such as proteins and DNA. However, lipids, especially PUFAs, are the ones most targeted by ROS [35]. Lipid oxidation products including hydroperoxides of phosphatidylcholine and cholesteryl ester have been detected and characterized in oxLDL and oxHDL [25]. Therefore, lipoproteins are the most likely targets of the lipid oxidation process in plasma. Indeed, when plasma was separated into two fractions, depending on the molecular size by membrane filtration (with a molecular weight cut-off at 100 kDa), almost all of the hydroperoxide-containing molecules were recovered in the high-molecular weight fraction.
The imbalance between circulating levels of lipoproteins (LDL and VLDL) relative to those of HDL is associated with atherogenesis. LDL is a major substrate for oxidation at the arterial wall, and the oxidatively modified form of LDL (oxLDL) is more important than native LDL in the process of atherogenesis [17,18,36,37]. However, oxHDL, but not oxLDL, is detected in plasma by the Fe-ROMs test. This observed uneven distribution of lipid hydroperoxides in plasma lipoproteins may be attributed to the following two possibilities: Firstly, a substantial amount of oxLDL is formed in the blood, but most of its oxidation products are rapidly metabolized into other molecules or removed by HDL. HDL, especially small, dense HDL3, has been reported to potently protect LDL against oxidative stress, and such potent antioxidative activity is related to the transfer of oxidized lipids from LDL to HDL [23,38]. Thus, HDL might function as a sink for oxidized lipids in the blood [39]. As a second possibility, HDL might be more susceptible to oxidation in the blood than LDL. Interestingly, lipid hydroperoxides were reported to accumulate more rapidly in HDL than in LDL in vivo, because of the action of LDL-associated antioxidants such as ubiquinol-10 [40]. As a consequence, LDL lipids are relatively peroxide-free.
Our HPLC analysis revealed that carriers of hydroperoxides were eluted at a position just after the majority of HDLs were eluted and coincidentally with very small HDL (Figure 5d). HDLs are a class of structurally and functionally heterogeneous particles. They are classified on the basis of their density and size, resulting in the large buoyant HDL2 and the small dense HDL3. HDL3c represents a minor sub-fraction of HDL3, accounting for about 6% of the total HDL mass, and its diameter is about 6.7 nm [35,41,42]. It was reported that HDLs, in particular the small dense HDL3, exert anti-inflammatory and antioxidant activities, suggesting that this HDL plays a role as a ROS scavenger in inflammatory sites [37]. Copper(II)-induced oxidation of LDL in the presence of total HDL, HDL2, or HDL3 demonstrated a less well-marked increase of hydroperoxides within LDL, but with a rapid production of hydroperoxides in HDL [43]. Further, HDL3 appeared to be more effective in preventing the hydroperoxidation of LDL. Taken together, our data indicate that it is likely that very small-sized HDL, for example, HDL3c, was strongly hydroperoxidized in the blood and thus detected by the Fe-ROMs test. However, a possibility still exists that HDL sub-fractions other than very small HDL are also hydroperoxidized in plasma, because the recovery of hydroperoxides after HPLC analysis was less than 20%.
HDL retards atherosclerosis via multiple mechanisms, including reverse cholesterol transport (RCT) [44,45], anti-inflammatory effects [46], and antioxidant ability [46]. However, in subjects with atherosclerosis, HDL becomes dysfunctional, which diminishes these anti-atherosclerotic functions. One of the main reasons for this loss of function is oxidative changes in HDL proteins and lipids. Various reports have described that the site-specific oxidation of apoA-1, the major HDL protein, by myeloperoxidase impairs RCT by the ATP-binding cassette transporter A1 pathway [47,48]. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls such as MDA and acrolein, which can be produced in the body through lipid peroxidation [49]. The antioxidant action of HDL is attributed to HDL-associated lipolytic enzymes such as paroxonase 1, as well as apoA-I [45,50]. Moreover, HDL particles protect against oxidative stress through their capacity to accept phospholipid hydroperoxides from LDL, and to reduce them to inactive hydroxides via the oxidation of methionine residues in apoA-I [23], indicating that the elevated peroxidation of lipids probably contributes to the antioxidant action. Collectively, these observations provide evidence that peroxidation of lipids in HDL is a potential mechanism for generating dysfunctional HDL.
This study showed that HDL was the principal carrier of lipid hydroperoxides in human plasma when the Fe-ROMs test was used for the determination of oxidative stress. This is an important finding because oxHDL, which is a risk factor for developing atherosclerosis, can be assayed by the Fe-ROMs test. Enzyme immunoassay kits have been developed for the detection and quantification of oxHDL in plasma or serum. In these assays, antibodies against the secondary oxidation products of HDL, such as MDA-HDL, have been used. However, these kits are not used routinely in a clinical examination setting because they require much labor and are expensive. On the other hand, our Fe-ROMs test is inexpensive and convenient for the determination of oxHDL. Our study indicated that hydroperoxidized HDL can be estimated from Fe-ROMs levels in plasma. However, as demonstrated by Bowry et al. [51], LDL carries lipid hydroperoxides, although their levels are far lower than those of HDL. Additionally, lipoproteins other than HDL may carry a substantial amount of hydroperoxides in subjects with cardiovascular disease or low antioxidant intake and in heavy smokers. Therefore, we recommend using plasma samples in a primary assay for the determination of oxHDL, and if necessary, supernatants after dextran sulfate-Mg 2+ precipitation in a secondary assay.

Subjects
Eleven healthy male subjects (aged 30.2 ± 14.3) and 10 male patients (age: 15.7 ± 12.1) who had experienced acute febrile disease were briefed about the study protocol and possible risks, signed the informed consent, and voluntarily participated in the present study. The study protocol was approved by the Ethics Committee of Kyoto Prefectural University of Medicine (RBMR-C-983, 9 November 2011). The patients were included in this study, because they showed a wide range of d-ROMs levels from normal to high. Blood samples were collected from the antecubital vein into sodium heparin-containing blood collection tube for the measurement of reactive oxygen metabolites. The blood samples were centrifuged at 4 • C immediately after collection, and the plasma was separated and stored at −80 • C until analysis could be performed.

The Measurement of Reactive Oxygen Metabolites by the d-ROMs and Fe-ROMs Tests
The amount of hydroperoxides present in a 20 µL volume of plasma was estimated by performing the d-ROMs test using the FRAS4 system according to the manufacturer's instructions (Wismerll Co., Tokyo, Japan). The results obtained from the d-ROMs test were expressed in arbitrary units called "Carratelli units" (U.CARR). In the Fe-ROMs test, 186 µL of 0.1 M acetate buffer (pH 4.8) containing 100 µM Fe(NH 4 ) 2 (SO 4 ) 2 or FeCl 3 was added into each well of a 96-well plate and warmed to 37 • C. Subsequently, 10 µL of 100 mM N,N -diethyl-p-phenylenediamine and an aliquot (4 µL) of plasma were added into each well of the 96-well plate and reacted at 37 • C. The reaction product was measured by using an xMark microplate spectrophotometer (Bio-Rad Laboratories, Inc.) with the wavelength, reading mode, and kinetic parameter set at 505 nm, kinetic, and rate, respectively. The results of the Fe-ROMs test were expressed as the rate of increase in OD (mOD/min) during a 3 min period from 2 to 5 min after the addition of the plasma, where 1 mOD/min corresponded to 23.7 µM tert-butyl hydroperoxide. Measurements were run in duplicate and a mean value was used in the analyses (intra-assay coefficient of variability = 3.84%).

Fractionation of HDL by the Dextran Sulfate-Mg 2+ Precipitation Method
The plasma was ultra-filtered by using a Millex-HV syringe filter unit (0.45 µm, PVDF membrane: polyvinylidene fluoride membrane). Part of the filtrate was stored at −80 • C as the plasma fraction, and the rest of it was processed to isolate the HDL fraction as reported previously [52]: 0.9 mL of the filtrate was mixed at room temperature with 0.1 ml of a solution (pH 7.3) containing 1% dextran sulfate sodium salt and 0.5 M MgCl 2 . The mixture was then stood at room temperature for 10 min and centrifuged (1500× g) for 30 min at 4 • C. The supernatant was ultra-filtered by passing through a 0.45 µm-Millex-HV syringe filter unit, and the filtrate was then stored as the HDL fraction at −80 • C. The stored plasma and HDL fractions were used for the lipoprotein analysis by using a dual detection HPLC system at Skylight Biotech (Akita, Japan) according to the procedure described by Usui et al. [53].

Lipoprotein Analysis of the Plasma by HPLC
Any turbidity in the plasma samples was cleared by ultrafiltration through a 0.45-µm Millex-HV syringe filter unit. The cleared plasma sample (0.5 mL) was then separated on an OH pak SB-804HQ column (300 × 8.0 mm) with 0.15 mol/L NaCl at a flow rate of 0.5 mL/min. The column temperature was set at 20 • C, and elution was monitored by absorbance at 260 nm. The column eluate was collected in 0.5 mL fractions and used for the Fe-ROMs test, HDL ELISA assay, and SDS-PAGE analysis.
The HDL ELISA assay was done by using an HDL Human ELISA Kit. Briefly, the column eluate (fractions 14-18) was diluted 1:1 with 1× Diluent M; and an aliquot (25 µL) of the diluted sample was added to the wells of a 96-well plate that had been pre-coated with an antibody specific for HDL. After this addition, the assay was done as indicated in the manufacturer's instructions; and HDL concentrations were calculated from the standard curve.

Protein Analysis by SDS-PAGE
An aliquot (5 µL) of the column eluate was heated at 70 • C for 10 min in a total 40 µL of a Bolt LDS sample Buffer containing Bolt Reducing Agent. It was then applied on a Bolt 4%-12% Bis-Tris Plus gel, and electrophoresed in Bolt MOPS SDS Running Buffer at 200 volts for 35 min. In parallel, 2 µL of the Bio-Rad Precision Plus Protein Dual Color Standards and 5 µL of 1 mg/mL human serum albumin were run. The gels were then washed with ultrapure water, stained for 1 h with Imperial Protein Stain (Thermo Fisher Scientific, Waltham, MA, USA), and destained with ultrapure water. The stained protein bands were detected by using an Image Quant LAS 4000 mini (GE Healthcare Life Sciences, Marlborough, MA, USA).

Protein G Affinity Chromatography
The plasma was cleared by ultra-filtration using a Millex-GV syringe filter unit (0.22 µm, PVDF). The cleared plasma (0.3 mL) was loaded onto a Protein G Sepharose 4 Fast Flow column (gel volume of 2 mL) and eluted with 10 mmol/L phosphate-buffered saline (pH 7.4) at 0.2 mL/min. The eluate was collected in a total of 18 fractions, each containing 0.2 mL. Seven fractions with high absorbance at 280 nm were collected and concentrated by using an Amicon Ultra-0.5 mL 100K (Merck KGaA, Darmstadt, Germany) according to the manufacturer's instruction, and analyzed by electrophoresis on a Bolt 4%-12% Bis-Tris Plus gel.

Other Methods
Quantitation of MDA-HDL: MDA-HDL was quantitated by using an OxiSelect Human Oxidized HDL ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA). For this assay, the HDL fraction was first isolated from the plasma by the dextran sulfate-Mg 2+ precipitation method. It was then diluted 1:50 with Assay Diluent, and an aliquot (100 µL) of the diluted HDL was used for the determination of MDA-HDL.
Purification of LDL/VLDL: One ml of plasma from a healthy male subject was used for the LDL/VLDL purification. The LDL/VLDL was purified by using an LDL/VLDL Purification Kit (Cell Biolabs, Inc.), and its activity was determined by performing the Fe-ROMs test after dialysis against a phosphate-buffered saline (pH 7.4).

Statistical Analysis
Data were presented as mean ± standard deviation (SD) and subjected to a one-way analysis of variance ANOVA with Tukey HSD post-hoc comparisons. The Pearson's correlation coefficient was used to identify the association between the levels obtained by the d-ROMs and Fe-ROMs tests. It was also conducted for the association between the levels quantified by the Fe-ROMs test and the MDA-HDL ELISA kit after the HDL fractionation. The analysis was performed using SPSS software version 24 (IBM Japan, Tokyo, Japan), and the statistical level of significance was set at 0.05.

Conclusions
The Fe-ROMs test was demonstrated to be able to assess oxidative stress with ease by using just a small amount of human plasma. Further, oxidized HDL can be estimated from Fe-ROMs levels in plasma. Emerging evidence shows that the anti-atherogenic role of HDL is not simply defined by the plasma HDL-C level but rather by a functional property of HDL. Because the functionality of HDL is affected by the oxidization of HDL components, the measurement of oxHDL must be useful for the assessment of the risk of cardiovascular disease.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/2/454/s1. Author Contributions: Fumiaki Ito and Tomoyuki Ito were involved in project conception, design, experimental procedures, and data analysis; Chinatsu Suzuki, Tomoyo Yahata, Kazuyuki Ikeda, and Kenji Hamaoka were involved in project design and patient recruitment; Fumiaki Ito wrote the paper.

Conflicts of Interest:
The authors declare no conflicts of interest.