Judah Folkman , a pioneer in the study of angiogenesis More than 30 years ago , Judah Folkman found a revolutionary new way to think about cancer . He postulated that in order to survive and grow , tumors require blood vessels , and that by cutting off that blood supply , a cancer could be starved into remission . What began as a revolutionary approach to cancer has evolved into one of the most exciting areas of scientific inquiry today . Over the years , Folkman and a growing team of researchers have isolated the proteins and unraveled the processes that regulate angiogenesis . Meanwhile , a new generation of angiogenesis research has emerged as well , widening the field into new areas of human disease and deepening it to examine the underlying biological processes responsible for those diseases . Early evidence of tumor cells releasing specific growth factor for blood vessels In 1939 , Ide et al. -LSB- 1 -RSB- were the first to suggest that tumors release specific factors capable of stimulating the growth of blood vessels . In 1945 , Algire and Chalkley -LSB- 2 -RSB- were the first to appreciate that growing malignancies could continuously elicit new capillary growth from the host . They used a transparent chamber implanted in a cat 's skin to study the vasoproliferative reaction secondary to a wound or implantation of normal or neoplastic tissues . They showed that the vasoproliferative response induced by tumor tissues was more substantial and earlier than that induced by normal tissues or following a wound . They concluded that the growth of a tumor is closely connected to the development of an intrinsic vascular network . In 1956 , Melwin and Algire -LSB- 3 -RSB- found that the vasoproliferative response of normal or neoplastic tissues transplanted into muscle was not significantly different with respect to the time of onset of new blood vessels , though it was stronger when the implantation was performed in a resection area . In addition , while normal tissues induced a vasoproliferative response confined to the host , tumor tissues induced the formation of neovessels that pierced the implant . Lastly , the intensity of the response seemed to be influenced by the distance between the implant and the host 's vessels : normal tissue was unable to induce a response if placed more than 50 μm away , whereas tumor tissue had a longer activity range . In 1968 , Greenblatt and Shubik -LSB- 4 -RSB- implanted Millipore chambers -LRB- pore size 0.45 μm -RRB- into a hamster 's cheek pouch and placed some tumor fragments around them . In a few days , the growing tumor mass engulfed the whole chamber , whose pores were permeable to the tumor interstitial fluid , but not to the tumor cells . New blood vessels , however , were formed in any case very likely through the release of a diffusible factor that could pass through the pores . Ehrman and Knoth -LSB- 5 -RSB- confirmed these data with tumor fragments laid on Millipore filters planted on the chick embryo chorioallantoic membrane -LRB- CAM -RRB- . Tumors in isolated perfused organs : absence of angiogenesis In 1963 , Folkman and Becker studied hemoglobin solutions as potential substitutes for blood transfusion . To test which solution was optimal for tissue survival , they perfused these solutions through the vasculature of canine thyroid glands , by using an apparatus with a silicone rubber oxygenator . The glands survived for about 2 weeks . They could distinguish different hemoglobin preparations by histologic analysis of the thyroid glands after a week or more of continuous arterial perfusion . To determine if these isolated organs could support growth , they injected them with adult mouse melanoma cells . Tiny tumors developed but stopped growing at 1 - to 2-mm diameter and never became vascularized -LSB- 6 -RSB- . Endothelial cells swelled and could not proliferate in the presence of free hemoglobin solutions lacking platelets -LSB- 7 -RSB- . However , the tumors were not dead . When they were transplanted to their host mice , they rapidly vascularized and grew to more than 1 cm3 . Folkman et al. observed that when tumor cells were inoculated into isolated perfused organs , tumors were limited in size to 1 -- 2 mm3 -LSB- 6 -RSB- . Subsequently , they found that neovascularization does not occur in isolated perfused organs and that tumors transplanted from these organs to syngeneic mice became vascularized and grew rapidly to 1 -- 2 mm3 . This was the first evidence that the absence of neovascularization correlated with severe restriction of tumor growth . The data were consistent with work from Harry Green , who had shown long before that growth of rabbit tumors transplanted into the anterior chamber of the guinea pig coincided with the growth of new blood vessels . Tumors that remained viable , but did not grow , had no visible blood vessels -LSB- 8 -RSB- . Hypothesis : tumor growth is angiogenesis dependent In 1971 , Folkman published in the `` New England Journal of Medicine '' a hypothesis that tumor growth is angiogenesis dependent and that inhibition of angiogenesis could be therapeutic -LSB- 9 -RSB- . This article also introduced the term anti-angiogenesis to mean the prevention of new vessel sprout from being recruited by a tumor . The hypothesis predicted that tumors would be enable to grow beyond a microscopic size of 1 -- 2 mm3 without continuous recruitment of new capillary blood vessels . This concept is now widely accepted because of supporting data from experimental studies and clinical observations carried out over the intervening years . Evidence that tumors are angiogenesis dependent Folkman and collaborators provide evidence for the dependence of tumor growth on neovascularization : Tumor growth in the avascular cornea proceeds slowly at a linear rate , but after vascularization , tumor growth is exponential -LSB- 10 -RSB- . Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye and observed for a period up to 6 weeks remain viable , avascular , and of limited size -LRB- less than 1 mm3 -RRB- and contain a population of viable and mitotically active tumor cells . These tumors induce neovascularization of the iris vessels but are too remote from these vessels to be invaded by them . After implantation contiguous to the iris , which had abundant blood vessels , the tumors induced neovascularization and grow rapidly , reaching 16,000 times the original size within 2 weeks -LSB- 11 -RSB- . This experiment introduced the concept of tumor dormancy brought about by prevention of neovascularization . In a parallel study , tumors were suspended in the aqueous humor of the anterior chamber , placed at various distances from the iris vessels , and compared with tumors implanted directly on the iris and with those implanted in the cornea -LSB- 12 -RSB- . Moving the distant , dormant tumors closer to the iris jump started their growth . This suggested that this type of tumor dormancy was caused not by cell cycle arrest or immune control , but by a lack of blood supply.B-16 mouse melanoma , V-79 Chinese hamster lung cells , and L-5178 Y murine leukemia cells were plated in soft agar -LSB- 13 -RSB- . After 6 -- 7 days of incubation , spheroid colonies of 0.1 mm were visible . All spheroids first enlarged exponentially for a few days and then continued on a linear growth curve for 5 -- 23 weeks before reaching a diameter beyond which there was no further expansion . This was termed the dormant phase . After the dormant diameter was reached , these spheroids remained viable for 3 -- 5 months , or as long as they were frequently transferred to new medium . Cells in the periphery of the spheroid incorporated 3H-labeled thymidine while cells in the center died . This is a form of population dormancy in which the proliferating cells near the surface of the spheroid just balance those dying cells deep in the center of the spheroid.Tumors implanted on the CAM of the chick embryo do not exceed a mean diameter of 0.93 ± 0.29 mm during the prevascular phase -LRB- approximately 72 h -RRB- . Rapid growth begins , however , within 24 h after vascularization , and tumors reach a mean diameter of 8.0 ± 2.5 mm by 7 days -LSB- 14 -RSB- . Tumors grown in the vitreous of the rabbit eye remain viable but attain diameters of less than 0.50 mm for as long as 100 days . Once such a tumor reaches the retinal surface , it becomes neovascularized and within 2 weeks can undergo a 19,000-fold increase in volume over the avascular tumor -LSB- 15 -RSB- . The CAM appears at day 5 during development of the chick embryo . The 3H-thymidine labeling index of its vascular endothelium decreases with age , with an abrupt reduction at day 11 -LSB- 16 -RSB- . Prior to day 11 , labeling index is approximately 23 % ; during 11 days , the labeling index decreases to 2.8 % , and subsequently , the cells begin to acquire the structural characteristics of matured , differentiated endothelium . One-millimeter fragments of fresh Walker 256 carcinoma were implanted on the CAM from day 3 to day 16 -LSB- 14 -RSB- . The size of the tumors was measured daily , and the onset of vascularization of each tumor was determined in vivo with a stereomicroscope and confirmed with histological sections . Proliferation of chick capillaries occurred in the neighborhood of the tumor graft by 24 h after implantation , but capillary sprouts did not penetrate the tumor graft until approximately 72 h . During the avascular phase , tumor diameter did not exceed 1 mm . Small tumor implants of 0.5 mm or less grew to 1 mm and stopped expanding . Larger tumor implants of 2 - or 3-mm shrank until they reached 1-mm diameter . During the first 24 h after penetration by capillaries , there was a rapid tumor growth . Neovascularization was not grossly observable with the stereomicroscope until after day 10 or 11 . Tumors implanted on the CAM after day 11 grow at slower rate in parallel with the reduced rates of endothelial growth.When tumor grafts of increasing size -LRB- from 1 to 4 mm -RRB- are implanted on the 9-day CAM , grafts larger than 1 mm undergo necrosis and autolysis during the 72-h prevascular phase . They shrink rapidly until the onset of neovascularization , when rapid tumor growth resumes -LSB- 14 -RSB- . In another study -LSB- 17 -RSB- , the behavior of tumor grafts on the CAM was compared to grafts of normal adult and embryonic tissues . In tumor tissue , preexisting blood vessels within the tumor graft disintegrated by 24 h after implantation . Neovascularization did not occur until after at least 3 days , and only by penetration of proliferating host vessels into the tumor tissue . There was marked neovascularization of host vessels in the neighborhood of the tumor graft . By contrast , in embryonic graft , preexisting vessels did not disintegrated . They reattached by anastomosis to the host vessels within 1 -- 2 days , but with minimal or almost no neovascularization on the part of the host vessels . In adult tissues , the preexisting graft vessels disintegrated ; there was no reattachment of their circulation with the host , and adult tissues did not stimulate capillary proliferation . These studies suggest that only tumor grafts are capable of stimulating formation of new blood vessels in the host.In transgenic mice that develop carcinomas of the β cells in the pancreatic islets , large tumors arise only from a subset of preneoplastic hyperplastic islets that have become vascularized -LSB- 18 -RSB- . Isolation of the first angiogenic tumor factor Until the early 1970s , it was widely assumed that tumors did not produce specific angiogenic proteins . The conventional widsom was that tumor vasculature was an inflammatory reaction to dying or necrotic tumor cells . Previous studies had shown that tumor-stimulated vessel growth did not require direct contact between tumor and host tissue -LSB- 4 , 5 -RSB- . This made sense to Folkman , who reasoned that a soluble factor would be more likely to reach nearly than distant blood vessels . He and his colleagues isolated an angiogenic factor in 1971 -LSB- 19 -RSB- . The homogenate of a Walker 256 carcinoma -- a breast tumor of Sprague-Dawley rats -- was fractionated by gel-filtration on Sephadex G-100 . The fraction that exhibited the strongest angiogenic activity had a molecular weight of about 10,000 daltons and consisted of 25 % RNA , 10 % proteins , and 58 % carbohydrates , plus a possible lipid residue . It was inactivated by digestion with pancreatic ribonuclease , or by heating at 56 °C for 1 h , and was not modified when kept at 4 °C for 3 months , nor when treated with trypsin for more than 3 days . This active fraction was subsequently called `` tumor angiogenesis factor '' -LRB- TAF -RRB- -LSB- 19 -RSB- . Both the cytoplasmic and the nuclear fractions of tumor cells stimulated angiogenesis . In the nuclear fraction , this was found to be associated with nonhistonic proteins -LSB- 20 -RSB- . Tumor angiogenesis factor has since been nondestructively extracted from several tumor cell lines , and several low molecular weight angiogenic factors have been isolated , again from the Walker 256 carcinoma . These factors induced a vasoproliferative response in vivo when tested on rabbit cornea or chick CAM , and in vitro on cultured endothelial cells -LSB- 21 -- 23 -RSB- . First evidence of the existence of the avascular and vascular phases of solid tumor growth The earliest evidence of the existence of the two phases was obtained by Folkman and collaborators in 1963 , who perfused the lobe of a thyroid gland with plasma and inoculated a suspension of melanoma B16 tumor cells through the perfusion fluid . These cells grew into small , clearly visible black nodules . The nodules did not exceed 1 mm in diameter and did not connect with the host 's vascular network . Their outer third generally remained vital , while the interior portion underwent necrosis . Reimplanted nodules , on the other hand , equipped themselves with a vascular network and grew very rapidly . The conclusion was thus drawn that the absence of vascularization limits the growth of solid tumors . Further research by Folkman 's group resulted in an experimental system in which the tumor , or its extracts , could be separated from the vascular bed -LSB- 24 , 25 -RSB- . This system was based on subcutaneous insufflation to lift the skin of a rat and form a poorly vascularized region below it . Millipore filters containing Walker 256 cancer cells or their cytoplasmic or nuclear extracts -LRB- TAF -RRB- were implanted into the fascial floor of the dorsal air sac . At intervals thereafter , 3H-labeled thymidine was injected into the air sac , and the tissues were examined by autoradiography and electron microscopy . Autoradiographs showed thymidine-3H labeling in endothelial cells of small vessels , 1 -- 3 mm from the site of implantation , as early as 6 -- 8 h after exposure to tumor cells . DNA synthesis by endothelium subsequently increased , and within 48 h new blood vessels formation was detected . The presence of labeled endothelial nuclei , endothelial mitosis , and regenerating endothelium was confirmed by electron microscopy . Tumor angiogenesis factor also induced neovascularization and endothelial DNA synthesis after 48 h. Further ultrastructural autoradiographic studies were carried out with the same model -LSB- 25 -RSB- . It was apparent that by 48 h there was ultrastructural evidence of regenerating endothelium , including marked increase in ribosomes and endoplasmic reticulum , scarce or absent pinocytotic vesicles , and discontinuous basement membrane . Labeled endothelial cells were seen along newly formed sprouts as well as in parent vessels . Furthermore , pericytes were also shown to synthesize DNA . In another series of experiments , 1-mm fragments from Brown-Pearce and V2 carcinomas were implanted into the avascular stroma of a rabbit cornea 1 -- 6 mm away from the limbic vessels , and observed the tumor growth daily with a stereomicroscope -LSB- 10 -RSB- . After 1 week , new blood vessels had invaded the cornea starting from the edge closer to the site of implantation and developed in that direction at 0.2 mm and then about 1 mm/day . Once the vessels reached the tumor , it grew very rapidly to permeate the entire globe within 4 weeks . Dormancy of micrometastases may be governed by angiogenesis Folkman and collaborators found that metastases were suppressed when a primary tumor was implanted and allowed to grow in nude mice , whereas they underwent neovascularization and became clinically evident when primary neoplasm was removed . In the absence of angiogenesis , micrometastases rarely exceeded 0.2-mm diameter and contained many proliferating tumor cells balanced by many apoptotic cells . When they were allowed to become angiogenic , they grew rapidly . Dormancy may be generalizable to a variety of tumors in which blocked angiogenesis results in balanced tumor cell proliferation and apoptosis -LSB- 26 -RSB- . Prognostic significance of tumor vascularity In 1972 , Brem in the Folkman laboratory reported the first quantitative method for histologic grading of tumor angiogenesis . He correlated neovascularization in human brain tumors with tumor grade -LSB- 27 -RSB- . In the early 1990s , Weidner and collaborators -LSB- 28 -- 30 -RSB- showed that measurement of microvascular density within isolated regions of high vessel concentration -LRB- i.e. , hotspots -RRB- was a prognostic indicator for human breast and prostate carcinomas . Microvascular density counting protocols have become the morphological gold standard to assess the neovasculature in human tumors . This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels . Microvascular density determined in primary tumors is significantly associated with metastasis and prognosis in several solid and hematological tumors . Antiangiogenesis The existence of specific angiogenesis inhibitors was first postulated by Folkman in 1971 in an editorial . No angiogenesis inhibitors existed before 1980 , and few scientists thought at that time that such molecules would ever be found . From 1980 to 2005 , Folkman 's laboratory reported the discovery of 12 angiogenesis inhibitors -LRB- Table 1 -RRB- . The first angiogenesis inhibitor was found in cartilage , an avascular tissue that resists invasion by many tumors -LSB- 31 -RSB- . Brem and Folkman demonstrated that tumor-induced vessels were inhibited by a diffusible factor from neonatal rabbit cartilage -LSB- 32 -RSB- . The partially purified inhibitor suppressed tumor growth when it was infused into the vascular bed of murine and rabbit tumors -LSB- 33 -RSB- . 2-methoxyestradiol was first reported by Fotsis et al. -LSB- 34 -RSB- , and the article reporting 2-methoxyoestradiol and its molecular mechanism as an inhibitor of tubulin polymerization by acting at the colchicine site was published a month later -LSB- 35 -RSB- . Table 1Angiogenesis inhibitors discovered in Folkman 's laboratory from 1980 to 20051980 . Interferon alpha-beta1982 . Platelet factor 4/protamine1985 . Angiostatic steroids1990 . Fumagillin1994 . Angiostatin1994 . Thalidomide1994 . 2-methoxyestradiol1997 . Endostatin1999 . Cleaved antithrombin III2002 . 3-Aminotholidomide2003 . DBF-maf2005 . Caplostatin Interferon alpha Interferon alpha was first shown to inhibit endothelial cell migration in a dose-dependent and reversible manner in 1980 by Zetter in Folkman 's laboratory -LSB- 36 -RSB- . Since 1988 , interferon alpha has been used successfully to cause complete and durable regression of life-threatening pulmonary hemangiomatosis , hemangiomatosis of the brain , airway and liver in infants , recurrent high-grade giant cell tumors refractory to conventional therapy , and angioblastomas -LSB- 37 -- 39 -RSB- . A 12-year-old boy with fatal pulmonary hemangiomatosis had a complete remission and recovered completely after 7 months of therapy with interferon alpha . Therapy was continued for 7 years . This led to the successful use of low-dose daily interferon alpha therapy administered subcutaneously to infants with sight-threatening or life-threatening hemangiomas and hemangioendotheliomas of the heart , airway , and liver -LSB- 37 -- 39 -RSB- . Platelet factor 4/protamine Protamine was shown to be an angiogenesis inhibitor -LSB- 40 -RSB- , but cumulative toxicity from prolonged administration and a narrow window of angiostatic efficacy prevented its consideration for clinical use . Platelet factor 4 was first tested for antiangiogenic activity because its method of binding and neutralizing heparin is similar to that of protamine -LSB- 40 -RSB- . Recombinant human platelet factor 4 -LRB- rHuPF4 -RRB- has been produced -LSB- 41 -RSB- . It specifically inhibited endothelial proliferation and migration in vitro -LSB- 42 -RSB- . The inhibitory activities are associated with the carboxy-terminal region of the molecule . The growth of human colon carcinoma in athymic mice , as well as the growth of murine melanoma , was markedly inhibited by intralesional injections , whereas tumor cells were completely insensitive to rHuPF4 in vitro at levels that inhibited normal endothelial cell proliferation . Systemic administration of rHuPF4 has so far been ineffective against tumor growth , perhaps because of rapid inactivation or clearance of the peptide . Angiostatic steroids Folkman had begun to use the CAM of the chick embryo to detect angiogenic activity in fractions being purified from tumor extracts . The addition of heparin increased the speed of development of the angiogenic reaction so that it could be read 1 -- 2 days later -LSB- 40 -RSB- . But one problem with this assay is that occasionally eggshell dust falls on the CAM and causes background inflammation . Folkman guessed that adding cortisone to the CAM might eliminate the irritation from the shell dust but not abolish the tumor-angiogenic reaction . As expected , cortisone alone prevented shell dust inflammation without interfering with angiogenesis induced by tumor extracts . The surprise was that when heparin and cortisone were added together , tumor angiogenesis was inhibited -LSB- 43 -RSB- . Furthermore , when this combination of heparin and steroid was suspended in a methylcellulose disk and implanted on the young -LRB- 6-day -RRB- CAM , growing capillaries regressed leaving in their place , 48 h later , an avascular zone . The antiangiogenic effect was specific for growing capillaries . Mature nongrowing capillaries in older membranes were unaffected . Nonanticoagulant heparin had the same effect . A hexasaccharide fragment with a molecular weight of approximately 16,000 was found to be the most potent inhibitor of angiogenesis -LRB- in the presence of a corticosteroid -RRB- . The combination of the heparin hexasaccharide fragment and cortisone also inhibited tumor-induced angiogenesis in the rabbit cornea . The regression of a growing vessel exposed to heparin-steroid combinations begins with endothelial cell rounding and is followed by cessation of endothelial proliferation , desquamation of endothelial cells , and retraction of the capillary sprout -LSB- 44 -RSB- . These events occur as 24 -- 48 h and are accompanied by dissolution of the basement membrane of the new capillary vessels . Fumagillin Fumagillin was found by Ingber in the Folkman laboratory to inhibit endothelial cell proliferation without causing endothelial cell apoptosis , when a tissue culture plate of endothelial cells became contaminated with a fungus Aspergillus fumigatus fresenius -LSB- 45 -RSB- . A conditioned medium from fungal cultures contained an inhibitor of endothelial cell proliferation and angiogenesis , which , upon purification , was found to be fumagillin , a polyene macrolide . When capillary endothelial cells were stimulated by fibroblast growth factor-2 -LRB- FGF-2 -RRB- , half-maximal inhibition was observed with fumagillin at 100 pg/ml . This antiproliferative effect appeared to be relatively specific for endothelial cells because inhibition of nonendothelial cells , including tumor cells , was observed at up to 1,000-fold higher concentrations . Scientists at Takeda Chemical Industries -LRB- Osaka , Japan -RRB- made a synthetic analogue of fumagillin , called TNP-470 , which inhibits endothelial proliferation in vitro at a concentration 3 logs lower than the concentration necessary to inhibit fibroblasts , and tumor cells . Angiostatin and endostatin They were discovered by M. O'Reilly in Folkman laboratory based on Folkman 's hypothesis of a mechanism to explain the phenomenon that surgical removal of certain tumors leads to rapid growth of remote metastases . This hypothesis said that if tumors produce both stimulators and inhibitors of angiogenesis , an excess of inhibitors could accumulate within an angiogenic tumor . In the circulation however , the ratio would be reversed . Angiogenesis inhibitors would increase relative to stimulators , because of rapid clearance of stimulators from the blood . Folkman formulated this hypothesis after reading Bouck 's first report in 1989 that the emergence of tumor angiogenesis was the result of a shift in balance between positive and negative regulators of angiogenesis in a tumor -LSB- 46 -RSB- . Bouck reported that the switch to angiogenesis during tumorigenesis of transformed hamster cells was associated with downregulation of an inhibitor of angiogenesis , thrombospondin . She suggested that the switch to the angiogenic phenotype could be the result of a shift in the net balance of positive and negative regulators of angiogenesis . In 1991 , O'Reilly began to screen a variety of transplantable murine tumors for their ability to suppress metastases . A Lewis lung carcinoma was the most efficient . When the metastasis-suppressing primary tumor was present in the dorsal subcutaneous position , microscopic lung metastases remained dormant at a diameter of less than 200 μm surrounding a preexisting microvessel , but revealed no new vessels . Within 5 days after surgical removal of the primary tumor , lung metastases became highly angiogenic and grew rapidly , killing their host by 15 days -LSB- 47 -RSB- . This striking evidence that primary tumor could suppress angiogenesis in its secondary metastases by a circulating inhibitor was further supported by the demonstration that a primary tumor could also suppress corneal angiogenesis by an implanted pellet of FGF-2 . O'Reilly then succeeded in purifying this inhibitor from the serum and urine of tumor-bearing animals . It was a 38-kD internal fragment identical in amino acid sequence to the first four kringle structures of plasminogen and it was named angiostatin . Angiostatin specifically inhibited the proliferation of growing vascular endothelial cells and had no effect on resting confluent endothelial cells or on other cell types , including smooth muscle cells , epithelial cells , fibroblasts and tumor cells . It also inhibited growth of primary tumors by up to 98 % -LSB- 48 -RSB- and was able to induce regression of large tumors -LRB- 1 -- 2 % of body weight -RRB- and maintain them at a microscopic dormant size . Based on the same rationale and strategy , O'Reilly isolated and purified another angiogenesis inhibitor from a murine hemangioendothelioma . This inhibitor , called endostatin -LSB- 49 -RSB- , is a 20-kD protein with an N-terminal amino acid sequence identical to the carboxyterminus of collagen XVIII . It was purified directly from tumor cell-conditioned medium . Endostatin is also a specific inhibitor of endothelial proliferation and has no effects on resting endothelial cells or on other cell types . It is slightly more potent than angiostatin and also causes regression of large tumors to a microscopic size . Thalidomide In 1994 , D'Amato in the Folkman laboratory reported that thalidomide is an angiogenic inhibitor -LSB- 50 -RSB- . Corneal neovascularization in rabbits induced by FGF-2 or VEGF was blocked by orally administered thalidomide . This activity of thalidomide was mainly the result of its direct effect on inhibiting new blood vessel formation and not on suppression of infiltrating host inflammatory cells . Histologic sections of the pretreated neovascularized corneas were virtually free of inflammatory cells . Thalidomide also inhibited corneal neovascularization in mice , but it was necessary to give the drug by the intraperitoneal route and at high doses , because mice do not metabolize thalidomide effectively . 2-Methoxyestradiol In 1994 , D'Amato in Folkman laboratory demonstrated that a metabolite of estradiol , 2-methoxyestradiol , inhibited angiogenesis in the chick CAM -LSB- 35 -RSB- . Moreover , since 2-methoxyestradiol causes mitotic perturbations , they examined its interactions with tubulin and showed that 2-methoxyestradiol bound to colchicine site of tubulin and , depending on reaction conditions , either inhibited assembly or seems to be incorporated into a polymer with altered stability properties . Cleaved antithrombin III A human small-cell lung carcinoma suppressed angiogenesis and tumor growth at remote sites in immunodeficient mice . These cells generated an enzyme in vitro that converted the 58-kD conformation of circulating antithrombin III to a 53-kD form of the protein -LSB- 51 -RSB- in which the externally configured stressed loop of antithrombin was retracted into the body of the molecule . The 53-kD form is a specific endothelial inhibitor and a potent angiogenesis inhibitor and has no thrombin binding activity . Caplostatin Caplostatin is a nontoxic synthetic analogue of fumagillin conjugated to a water-soluble-N - -LRB- 2-hydroxypropyl -RRB- methacrylamide -LRB- HPMA -RRB- copolymer -LSB- 52 , 53 -RSB- . Caplostatin has a similar broad antitumor spectrum of TNP-470 without any toxicity . In addition to its antiangiogenic activity , caplostatin is the most potent known inhibitor of vascular permeability -LSB- 53 -RSB- . Caplostatin prevents vascular leakage induced by VEGF , bradykinin , histamine , and platelet-activating factor and prevents pulmonary edema induced by interleukin-2 . Antiangiogenic chemotherapy Browder in the Folkman laboratory was the first to demonstrate the concept that by optimizing the dosing schedule of conventional cytotoxic chemotherapy to achieve more sustained apoptosis of endothelial cells in the vascular bed of a tumor , it is possible to achieve more effective control of tumor growth in mice , even if the tumor cells are drug resistant -LSB- 54 -RSB- . Conventional chemotherapy is administered at maximum tolerated doses followed by off-therapy intervals of 2 -- 3 weeks to allow the bone marrow and gastrointestinal tract to recover . In contrast , antiangiogenic chemotherapy is administered more frequently at lower doses , without long interruptions in therapy and with little or no toxicity . During antiangiogenic chemotherapy , endothelial cell apoptosis and capillary dropout precede the death of tumor cells that surround each capillary . Concluding remarks Currently , several compounds with angiostatic activity are approved for clinical use , and many are in late-stage clinical development . However , the results from clinical trials have not shown the antitumor effects which were expected following preclinical studies . It appears that clinical applications of antiangiogenic therapy are more complex than originally thought . The main problem in the development of antiangiogenic agents is that multiple angiogenic molecules may be produced by tumors , and tumors at different stages of development may depend on different angiogenic factors for their blood supply . Therefore , blocking a single angiogenic molecule was expected to have little or no impact on tumor growth . Current development of targeted antiangiogenic agents include their use in adjuvant settings and the combination of different antiangiogenic inhibitors to take a more comprehensive approach in blocking tumor angiogenesis . Advancing insights into fundamental mechanisms will be necessary in the development of novel anticancer strategies based on inhibition of angiogenesis .

O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O


An on-line post-column detection system for the detection of reactive-oxygen-species-producing compounds and antioxidants in mixtures Reactive oxygen species -LRB- ROS -RRB- can damage proteins , cause lipid peroxidation , and react with DNA , ultimately resulting in harmful effects . Antioxidants constitute one of the defense systems used to neutralize pro-oxidants . Since pro-oxidants and antioxidants are found ubiquitously in nature , pro-and antioxidant effects of individual compounds and of mixtures receive much attention in scientific research . A major bottleneck in these studies , however , is the identification of the individual pro-oxidants and antioxidants in mixtures . Here , we describe the development and validation of an on-line post-column biochemical detection system for ROS-producing compounds and antioxidants in mixtures . Inclusion of cytochrome P450s and cytochrome P450 reductase also permitted the screening of compounds that need bioactivation to exert their ROS-producing properties . This pro-oxidant and antioxidant detection system was integrated on-line with gradient HPLC . The resulting high-resolution screening technology was able to separate mixtures of ROS-producing compounds and antioxidants , allowing each species to be characterized rapidly and sensitively . Introduction The potentially toxic and beneficial properties of pro-oxidants and antioxidants have made them the focus of many studies . Pro-oxidants may represent a threat to health , whereas antioxidants may counteract these effects by scavenging pro-oxidants -LSB- 1 , 2 -RSB- . Antioxidants are very important in industrial processes as well as in biological systems . They are known to possess anti-inflammatory -LSB- 3 -RSB- , anti-cardiovascular disease -LSB- 4 -RSB- , antineurogenerative -LSB- 5 -RSB- , and anticancer properties -LSB- 6 -RSB- . Imbalances between pro-oxidants and antioxidants in favor of the pro-oxidants may result in oxidative stress , which in turn may result in oxidative damage -LSB- 7 -RSB- of cellular components in the form of lipid peroxidation , protein denaturation or DNA conjugation -LSB- 8 -RSB- . Oxidative stress has been associated with many diseases like cancer -LSB- 6 -RSB- , post-ischemic and neural degradation -LSB- 2 -RSB- , Parkinson 's and Alzheimer disease -LSB- 5 -RSB- , AIDS -LSB- 9 -RSB- , and aging and cardiovascular diseases -LSB- 4 -RSB- . The metabolism of pro-oxidants by cytochrome P450s is another important process that can result in the formation of reactive oxygen species -LRB- ROS -RRB- -LSB- 10 , 11 -RSB- . Because antioxidants may neutralize the potentially harmful effects of direct pro-oxidants or pro-oxidants formed upon bioactivation , much of the work done in this field has been directed at synthesizing antioxidants , in some cases organ - or tissue-specific ones -LSB- 12 -- 14 -RSB- . In addition , the food industry pays a great deal of attention to antioxidants in foods . Phenols in tea -LSB- 15 -RSB- , fish oils -LSB- 16 -RSB- , curcumin in curry -LSB- 17 -RSB- and flavanoids in plants -LSB- 18 -RSB- are only a few examples of the large number of natural compounds that have been and are being studied for their positive , antioxidant-based , effects . When the pro-oxidant or antioxidant effects of natural extracts or synthetic compounds are investigated , batch assay formats are normally utilized -LSB- 19 -- 21 -RSB- . On-line assays that measure the pro-oxidant or antioxidant effects of compounds are also described -LSB- 22 -- 24 -RSB- . However , when individual compounds in mixtures need be analyzed for their pro-oxidant or antioxidant properties using these techniques , cumbersome gradient HPLC separations are required before the purified compounds can be characterized . Moreover , care must be taken to ensure that the purified compounds are not oxidized or degraded in air before analysis . One strategy that could be employed to circumvent the need for this cumbersome purification and subsequent screening process is high-resolution screening -LRB- HRS -RRB- , which stands for `` on-line post-column biodetection after HPLC separation . '' HRS methodologies , which screen individual compounds in mixtures for affinity , have been developed for receptors -LRB- e.g. , the estrogen receptor -LSB- 25 -RSB- -RRB- , enzymes -LRB- e.g. , cathepsin B -LSB- 26 -RSB- , cytochrome P450s -LSB- 27 -RSB- -RRB- , and antibodies -LRB- e.g. , digoxin antibodies -LSB- 28 -RSB- -RRB- . These HRS strategies have proven to be very useful for the rapid profiling and identifying of individual ligands in active mixtures , especially when HRS systems are run simultaneously with MS -LSB- 29 -RSB- . Moreover , the use of these fast HRS strategies can result in a reduced risk of the oxidation or degradation of sample components before analysis . This paper presents the development and validation of a HRS-based on-line post-column detection system for the detection of ROS-producing compounds as well as antioxidants in mixtures . This so-called pro-oxidant and antioxidant detection -LRB- PAD -RRB- system is based on the oxidation of 4-hydroxyphenylacetic acid -LRB- 4HPAA -RRB- by H2O2 in the presence of horseradish peroxidase -LRB- HRP -RRB- to a highly fluorescent dimer -LSB- 30 , 31 -RSB- . Scheme 1 shows the general principles of the assay . H2O2 may be present as such or it may be formed from superoxide anion radicals -LRB- resulting from ROS-producing compounds -RRB- in the presence of superoxide dismutase -LRB- SOD -RRB- . After optimization , the on-line detection system was validated with the well-known ROS-producing compounds paraquat , menadione and duroquinone , and the antioxidants L-ascorbic acid and glutathione , in flow injection analysis -LRB- FIA -RRB- mode . Finally , the on-line PAD system was coupled to gradient HPLC and thus used in HRS mode to screen individual compounds in mixtures for their ROS-producing and/or antioxidant properties . Scheme 1Schematic representation of the principles of the biochemical assay Experimental section Materials L-ascorbic acid , L-glutathione -LRB- reduced ; GSH -RRB- and 4-hydroxyphenylacetic acid -LRB- 4-HPAA -RRB- were purchased from Aldrich -LRB- Zwijndrecht , The Netherlands -RRB- . Tween 20 , menadione , polyethyleneglycol 6000 -LRB- PEG6000 -RRB- , polyethyleneglycol 3350 -LRB- PEG3350 -RRB- , methylviologen -LRB- paraquat dichloride -RRB- , peroxidase -LRB- horseradish , type I ; HRP -RRB- , and superoxide dismutase -LRB- from bovine erythrocytes ; SOD -RRB- were purchased from Sigma -LRB- Zwijndrecht , The Netherlands -RRB- . β-Nicotinamide adenine dinucleotide phosphate tetra sodium salt -LRB- NADPH -RRB- was from Applichem -LRB- Lokeren , Belgium -RRB- . Methanol -LRB- MeOH -RRB- and isopropanol -LRB- IPA -RRB- were purchased from Riedel de Haën -LRB- Seelze , Germany -RRB- . Acetonitrile -LRB- ACN -RRB- was from Baker -LRB- Deventer , The Netherlands -RRB- . The MeOH , ACN and IPA were of HPLC reagent grade . Rat liver microsomes -LRB- β-NF induced -RRB- were prepared as described elsewhere -LSB- 32 -RSB- . In short : livers from β-NF induced rats were homogenized at 4 °C in two volumes of 50 mM potassium phosphate buffer -LRB- pH 7.4 -RRB- with 0.9 % sodium chloride using a Potter-Elvehjem -LRB- Sigma -RRB- homogenizer . The homogenate was centrifuged for 20 min at 12.000 × g , and the supernatant obtained was further centrifuged for 60 min at 100.000 × g . The resulting pellet was washed twice and subsequently resuspended in 50 mM potassium phosphate buffer -LRB- pH 7.4 -RRB- , 0.9 % sodium chloride , and 25 % glycerol , and stored at − 80 °C . The protein concentration in the microsomes was determined as 13.1 mg/ml . Protein concentrations were determined with the standard Bio-Rad -LRB- Hercules , CA , USA -RRB- protein assay based on the method of Bradford . Instrumentation A Gilson 234 auto injector -LRB- Villiers-le-Bel , France -RRB- equipped with a Rheodyne -LRB- Bensheim , Germany -RRB- six-port injection valve -LRB- injection loop , 50 μl -RRB- was used for sample injections . A Knauer K-500 HPLC pump -LRB- Berlin , Germany -RRB- was used to deliver the injected samples into the on-line PAD system in FIA mode . Two Knauer K-500 HPLC pumps were used to deliver the cofactors , substrate and enzymes by means of superloops -LRB- SL-A and SL-B -RRB- -LRB- 50 ml , Pharmacia , Peapack , NJ , USA -RRB- , which were kept on ice , into the PAD system . Prior to detection via an Agilent 1100 -LRB- Waldbronn , Germany -RRB- series fluorescence detector -LRB- λex 320 nm ; λem 409 nm -RRB- , a knitted reaction coil -LRB- 0.25 mm i.d. ; 1.59 mm o.d. ; internal volume of 75 μl -RRB- positioned in a Shimadzu CTO-10AC column oven -LRB- Duisburg , Germany -RRB- , was used to perform the enzymatic reaction on-line . To reduce pump pulsing , flow restrictors were inserted between the pumps and the superloops . The flow restrictors were made in a similar way to the ones used by Kool et al. -LSB- 27 -RSB- . Pro-oxidant and antioxidant assay optimization The initial optimization of the biochemical assay for the PAD system was performed off-line on a Shimadzu RF-1501 spectrofluorometer -LRB- λex 320 nm ; λem 409 nm -RRB- , before the biochemical assay was transferred to the on-line format for further optimization . All measurements were performed in triplicate at 37 °C in quartz cuvettes with total volumes of 2 ml . Different concentrations of enzymes -LRB- rat liver microsomal cytochrome P450s and cytochrome P450 reductase , SOD and HRP -RRB- , cofactor NADPH , and 4-HPAA were tested . Blocking reagents and detergents -LRB- that can improve the resolution of the PAD system -RRB- -LSB- 27 -RSB- and organic modifiers -LRB- that are necessary when the PAD system is operated in on-line gradient HPLC mode -RRB- were investigated as well . Potassium phosphate buffer -LRB- 50 mM ; pH 7.8 -RRB- was used in all experiments . Initial conditions were rat liver microsomes -LRB- 50 μg/ml -RRB- , NADPH -LRB- 40 μM -RRB- , 4-HPAA -LRB- 1 mM -RRB- , SOD -LRB- 10 U/ml -RRB- , and HRP -LRB- 10 U/ml -RRB- . Reactions were started after 5 min of pre-incubation with the addition of paraquat -LRB- 70 μM -RRB- . The different parameters were optimized in the abovementioned order . When the optimal -LRB- or best compromise -RRB- concentration of every parameter was subsequently used in the optimization of the next parameter . After the optimization process , the optimized conditions were used in the PAD system in FIA and HPLC mode . Pro-oxidant and antioxidant detection system in flow-injection analysis mode A schematic view of a similar on-line detection system to the PAD system used in FIA mode is shown and described elsewhere -LSB- 27 -RSB- . The main difference between the PAD system used in FIA mode and that used in HPLC mode is the replacement of a carrier solution used in FIA mode with a gradient reversed-phase HPLC system . The general scheme for a PAD system coupled on-line to gradient reversed-phase HPLC -LRB- described in the next paragraph -RRB- is shown in Fig. 1 , which also shows the schematics of the PAD system in general . The continuous mixing of enzymes -LRB- cytochrome P450s and cytochrome P450 reductase , HRP and SOD -RRB- from SL-A and cofactors/substrate -LRB- NADPH/4-HPAA -RRB- from SL-B with a carrier solution in a reaction coil is the basic principle of the PAD system -LRB- when used in FIA mode -RRB- . After mixing in a knitted reaction coil , ROS formed from pro-oxidants -LRB- after cytochrome P450/cytochrome P450 reductase-mediated bioactivation -RRB- are converted by SOD to -LRB- relatively stable -RRB- H2O2 . The subsequent H2O2-dependent conversion of non-fluorescent 4-HPAA to its fluorescent dimer by HRP yields a spectroscopic handle for the efficient measurement of ROS formation . Thus , the ROS formed by eluting compounds give rise to a temporary increase in the formation of fluorescent product , which is seen in the PAD system as a peak . When SL-B also contains a ROS-producing compound , continuous oxidation of the 4-HPAA results in an elevated fluorescent baseline . In this situation , injection of antioxidants causes a temporary decrease in 4-HPAA oxidation , resulting in a negative peak in the PAD system . Thus , the system is sensitive to both ROS-producing compounds and antioxidants . Fig. 1Schematic view of the PAD system used in HPLC mode . Superloop A -LRB- SL-A -RRB- and superloop B -LRB- SL-B -RRB- are used to deliver enzymes and substrates to the reaction coil , respectively . ROS-producing pro-oxidants and antioxidants are introduced into the system by a gradient reversed-phase HPLC system . Antioxidants and ROS-producing compounds temporarily alter the amount of fluorescent product formed , and this change is detected with a fluorescence -LRB- FLD -RRB- detector . After HPLC , the make-up pumps produce a counteracting gradient , resulting in a PAD-compatible constant organic modifier concentration . The effluent is then split 1:9 -LRB- 90 % to UV detection and 10 % to CYP EAD -RRB- . AS , autosampler Liquid chromatography coupled to the pro-oxidant and antioxidant detection system A general scheme for the PAD system coupled on-line to gradient reversed-phase HPLC is shown in Fig. 1 . Gradient HPLC separations were performed using a 30 mm length × 2 mm i.d. stainless steel column -LRB- Luna 3 μ C18 -LRB- 2 -RRB- , Phenomenex , Torrance , CA , USA -RRB- . When the PAD system was operated in HPLC mode , two pumps were used to control the LC gradient and two pumps were used directly after the HPLC column to compensate for the increased concentration of organic modifier -LRB- during the gradient -RRB- before the delivery of the effluent to the PAD system . The following gradient was used for the HPLC separations : an initial flow rate of 300 μl/min for 3 min at H2O : MeOH -LRB- 95:5 -RRB- ; a decreasie in the flow rate gradient for 6 min to 150 μl/min H2O : MeOH -LRB- 5:95 -RRB- ; 14 min with H2O : MeOH -LRB- 5:95 -RRB- at a flow rate of 150 μl/min . Thereafter , the column was re-equilibrated to the starting conditions in 5 min . To maintain a constant concentration of MeOH after the HPLC column , a second gradient with an increasing flow-rate was included in the system , after HPLC separation , with an initial flow-rate of 700 μl/min H2O : MeOH -LRB- 4:1 -RRB- for 4.5 min . An increase in the flow-rate gradient for 6 min to 850 μl/min H2O : MeOH -LRB- 100:0 -RRB- was then followed by a post-gradient flow-rate of 850 μl/min H2O : MeOH -LRB- 100:0 -RRB- for 14 min . Finally , re-equilibration to starting conditions was performed in 5 min . The H2O and MeOH of the increasing flow-rate gradient contained 100 mg/L Tween 20 . The final constant flow-rate was 1000 μl/min , with a MeOH concentration of 15 % in H2O . This flow was connected to a T-piece and split 1/9 with a flow splitter . 90 % was directed to the UV detector , while10 % was pumped into the PAD system . For HPLC analysis , all tested compounds were dissolved in 30 % MeOH in water . Results and discussion The primary aim of this study was to develop and validate a HRS-based on-line post-column detection system for the detection of ROS-producing compounds and antioxidants in mixtures . The biochemical assay is based on the oxidation of 4-hydroxyphenylacetic acid -LRB- 4HPAA -RRB- to a highly fluorescent dimer -LRB- see Scheme 1 -RRB- . After optimization , the on-line detection system was validated using the well-known ROS-producing compounds paraquat , menadione , and duroquinone , as well as the antioxidants L-ascorbic acid and glutathione , in flow injection analysis -LRB- FIA -RRB- mode . The on-line PAD system was then coupled to gradient HPLC and this was used in HRS mode to screen for individual compounds in mixtures based on their ROS-producing and/or antioxidant properties . Optimization of pro-oxidant and antioxidant detection assay The optimization of the biochemical assay for the PAD system was conducted first in an off-line batch format before the optimized biochemical assay was transferred to the PAD system in FIA and HPLC modes . Paraquat was used as a model ROS-producing compound . The following parameters were optimized : enzyme concentrations -LRB- rat liver microsomes , SOD , and HRP -RRB- , cofactor NADPH and substrate 4-HPAA , blocking reagents PEG3350 and PEG6000 , the detergent Tween 20 -LRB- which can improve the resolution of the on-line PAD system -RRB- and organic modifiers -LRB- that are necessary when the on-line PAD system is operated on-line in gradient HPLC mode -RRB- . All optimizations were performed without the presence of paraquat as the continuous ROS-producing compound in SL-B . However , paraquat -LRB- 0.036 mM -RRB- was added to the SL-B in the final optimized system in order to permit measurements of both pro-oxidants and antioxidants using the on-line PAD system . First , the concentration of cytochrome P450s/cytochrome P450 reductase-containing rat liver microsomes was evaluated . Insertion of this important , mainly hepatic , biotransformation system permits the reductive bioactivation of compounds that need to be bioactivated before they can exhibit their ROS-producing effects -LRB- e.g. , paraquat -LSB- 33 -RSB- -RRB- . For the liver microsomes , it was found that higher concentrations resulted in an increase in fluorescence signals up to at least 150 μg/ml -LRB- microsomal protein concentration -RRB- . This is due to increased cytochrome P450s/cytochrome P450 reductase-mediated redox cycling -LSB- 33 -RSB- . Since the use of high concentrations of rat liver microsomes increases the risk that on-line biochemical detection systems will become clogged -LSB- 27 -RSB- , a concentration of 50 μg/ml rat liver microsomes was used in subsequent experiments . For SOD , concentrations higher than 14 U/ml did not increase the fluorescence signal significantly . Therefore , this SOD concentration was used in subsequent experiments . For HRP , the same effect was observed at a concentration of 18 U/ml , and so this concentration was employed from then on . Increasing the concentration of 4-HPAA led to the increased formation of fluorescent dimer , up to a concentration of 1.2 mM , after which the additional increase in sensitivity was counteracted by an increase in noise . A 4-HPAA concentration of 1.2 mM was therefore used in later experiments . An NADPH concentration of greater than 44 μM did not increase the assay performance significantly and so this concentration was adopted . For the polymers PEG3350 and PEG6000 , concentrations of up to 5 mg/ml did not produce significant differences in assay performance . Since a PEG6000 concentration of 1 mg/ml is sufficient to reduce possible peak broadening in on-line biodetection systems -LSB- 27 -RSB- , this concentration was in subsequent experiments . Detergents , such as Tween 20 , can be used to prevent -LRB- membrane-bound -RRB- enzymes from adhering to reaction coils in on-line biodetection systems , thereby resulting in reduced peak broadening -LSB- 27 -RSB- . Solubilization of membrane-bound enzymes , such as cytochrome P450s , can occur at high detergent concentrations and will inactivate the microsomes under the present conditions . A Tween 20 concentration of 100 -- 200 mg/l showed minimal -LRB- 5 -- 20 % -RRB- enzyme denaturation . These Tween 20 concentrations are known to efficiently reduce peak broadening when used in cytochrome-P450-containing on-line biodetection systems -LSB- 27 -RSB- . A Tween 20 concentration of 100 mg/l was therefore used from then on . Organic modifiers may be useful for preventing enzymes and lipophilic compounds from adhering to the walls of reaction coils , thus preventing peak broadening -LSB- 27 -RSB- . Moreover , when on-line biodetection systems are operated in the HPLC mode , organic modifiers are automatically introduced via the HPLC gradient . When testing MeOH , ACN , and IPA in the off-line biochemical batch assay format , they were tolerated up to concentrations of 10 % , resulting in approximately half the fluorescence signal compared to that obtained when organic modifiers were not used . Optimized conditions were derived from the abovementioned experiments performed in the off-line batch assay format and subsequently translated to the on-line PAD system in FIA mode . These final conditions were : a carrier solution consisting of 10 % MeOH and 100 mg/l Tween 20 at a flow rate of 100 μl/min ; SL-A containing potassium phosphate buffer -LRB- 50 mM ; pH 7.8 -RRB- , rat liver microsomes -LRB- 50 μg/ml -RRB- , HRP -LRB- 18 U/ml -RRB- , and SOD -LRB- 14 U/ml -RRB- , and SL-B with the same buffer containing PEG6000 -LRB- 1 mg/ml -RRB- , NADPH -LRB- 44 μM -RRB- , and 4-HPAA -LRB- 1.2 mM -RRB- . For continuous ROS production -LRB- resulting in a stable fluorescent baseline -RRB- , paraquat was present in the optimized system in SL-B at a concentration of 0.036 mM . Both superloops had a flow-rate of 100 μl/min . PAD system in flow-injection analysis mode Before coupling the on-line PAD system to gradient HPLC , it was first evaluated and validated in FIA mode . Evaluation of the PAD system was done with three well-known model ROS-producing compounds -LRB- i.e. , paraquat , menadione , and duroquinone -RRB- , and two well-known model antioxidants -LRB- L-ascorbic acid and glutathione -RRB- . First , paraquat was injected in triplicate at different concentrations -LRB- 1.0 , 0.25 , 0.06 , 0.015 , 0.004 , 0.001 , 0 mM -RRB- into the PAD system in FIA mode . Figure 2a shows the resulting signals . The highest concentration of paraquat resulted in fluorescence quenching . An analogous FIA trace for the redox cycling compound menadione is depicted in Fig. 2b . Fig. 2Triplicate injections of a series of dilutions -LRB- dilution factor of 4 -RRB- of different compounds into the PAD system used in FIA mode . a The pro-oxidant paraquat -LRB- starting with 1.0 mM -RRB- . b The pro-oxidant menadione -LRB- starting with 0.15 mM -RRB- . c The antioxidant ascorbic acid -LRB- starting with 1.0 mM -RRB- . d The antioxidant glutathione -LRB- starting with 25 mM -RRB- Figure 2c and d show the resulting PAD traces when L-ascorbic acid and glutathione -LRB- well-known antioxidants -RRB- were injected in triplicate in a dose-response manner , respectively . It was shown that ascorbic acid acted as a pro-oxidant compound at low concentrations -LRB- Fig. 2c -RRB- . Pro-oxidant effects of ascorbic acid at low concentrations were previously demonstrated by Abudu et al. -LSB- 34 -RSB- , who stated that ascorbic acid can undergo one-electron reduction to form an ascorbyl radical . Ascorbic acid can also switch from being an antioxidant to being a pro-oxidant in the presence of transition metals -LSB- 35 -RSB- . The direct antioxidant effect of L-ascorbic was also shown using the PAD system -LRB- without paraquat in SL-B -RRB- by first injecting paraquat and then injecting a mixture of paraquat and L-ascorbic acid . Figure 3 shows that the addition of L-ascorbic acid neutralizes the ROS formed from paraquat . The same effect is shown for menadione -LRB- Fig. 3 -RRB- . Fig. 3Injections -LRB- triplicates -RRB- of different compounds into the PAD system in FIA mode -LRB- without the continuous addition of a pro-oxidant -RRB- : 1 -RRB- paraquat -LRB- 0.05 mM -RRB- ; 2 -RRB- paraquat -LRB- 0.05 mM -RRB- and ascorbic acid -LRB- 0.1 mM -RRB- ; 3 -RRB- menadione -LRB- 0.03 mM -RRB- ; 4 -RRB- menadione -LRB- 0.03 mM -RRB- and ascorbic acid -LRB- 0.1 mM -RRB- To determine the effects of the test compounds paraquat , menadione , L-ascorbic acid , and glutathione on the fluorescence baseline , injections of different concentrations of the test compounds into the PAD system were performed without HRP and SOD in SL-A . Menadione and duroquinone caused fluorescence signals when injected in concentrations larger then 0.8 and 0.4 mM , respectively . This implies that these compounds can only be measured efficiently at these or higher concentrations if a parallel signal measuring their auto-fluorescence is employed , so that this can be subtracted from the PAD signal . In order to determine the relative pro-oxidant or antioxidant effects of the compounds , and to compare the off-line batch assay and the on-line assay -LRB- in FIA and HPLC mode -RRB- with each other , the fluorescence signals per mole of the test compound were determined from the data obtained in the off-line batch assay and the PAD system in FIA and HPLC modes -LRB- Table 1 -RRB- . When comparing the relative fluorescence signals per mole of test compound obtained using the off-line batch and both of the on-line systems , the relative fluorescence signals appeared to differ significantly for some compounds . This difference might be explained by the pre-incubation step , which was only performed for the off-line batch assays and not for the on-line PAD measurements . In contrast , for the off-line batch assay and both of the on-line PAD system formats , the measured pro-oxidant or antioxidant effects were comparable for all test compounds in terms of determinations of their individual pro-oxidant or antioxidant effects . Although the results obtained using the off-line batch format and both on-line formats were not similar , the comparable results still allowed the off-line batch assay to be transformed into both on-line assay formats . We can expect these relatively complex assay systems to give slightly different assay characteristics when performed under different assay conditions , like off-line batch or on-line assay conditions . Therefore , we can conclude that the off-line batch assay format can be transferred to the present PAD system used to screen for antioxidants and ROS-producing compounds . Table 1Initial relative increases or decreases -LRB- in fluorescence units , FU -RRB- of different ROS-producing pro-oxidant compounds and antioxidants compared to paraquat for the PAD system used in FIA mode , in gradient HPLC mode and for a traditional batch assayPro-oxidant/AntioxidantFIA PAD system -LRB- FU/mol ± SEM -RRB- Detection limit for the FIA PAD system -LRB- nmol -RRB- HPLC PAD system -LRB- FU/mol ± SEM -RRB- Detection limit for the HPLC PAD system -LRB- nmol -RRB- Batch assay set-up -LRB- FU/mol ± SEM -RRB- Paraquat1 .00 ± 0.080.071.00 ± 0.330.91.00 ± 0.05 Menadione1 .55 ± 0.200.010.56 ± 0.010.41.60 ± 0.33 Duroquinone0 .13 ± 0.020.040.36 ± 0.191.30.16 ± 0.07Glutathione-2.20 ± 0.031.9-0.67 ± 0.188.0-1.01 ± 0.04 Ascorbic acid-4.58 ± 0.220.1-1.35 ± 0.130.2-1.22 ± 0.12 The detection limits of the PAD system used in the FIA and HPLC modes are also given Interday and intra-day variabilities were determined in the on-line PAD system in FIA mode as follows : intraday variability was determined by injecting paraquat -LRB- 0.2 mM -RRB- in triplicate at 3.5-hour time intervals into the PAD system . The intraday variability was determined without changing the contents of the superloops . For the interday variability , paraquat -LRB- 0.2 mM -RRB- was injected daily in triplicate for three days with fresh solutions in the superloops each day . Intraday variability was 3.3 % ± 1.1 % and interday variability 4.1 % ± 0.6 % , which are both within the ranges exhibited by bioanalytical screening methods -LSB- 27 , 29 -RSB- . Detection limits -LRB- Table 1 -RRB- were determined by triplicate injections of a series of dilutions of every test compound . The detection limit was defined as the concentration of test compound that gave an average signal of three times the noise -LRB- S/N ratio = 3 -RRB- . The sensitivities obtained for the different compounds , which are also intrinsically determined by their pro-oxidant or antioxidant potencies , are also indicated in Table 1 . These sensitivities were of the same order as those obtained with the off-line batch assay format -LRB- data not shown -RRB- . Thus , the present PAD system provided a useful novel rapid screening tool for pro-oxidant and antioxidant activities of individual compounds in mixtures in this respect too . On-line coupling of the PAD system to gradient HPLC The PAD system in gradient HPLC mode was evaluated by analyzing the five test compounds after HPLC separation with a decreasing flow-rate gradient . The advantage of this decreasing flow-rate gradient lies with the initially high flow-rate through the column at low concentrations of organic modifier , which results in better eluting compound resolution at the start of the gradient . At higher concentrations of organic modifier , the flow rates are obviously gradually decreased -LRB- and the post-column counteracting flow rates are gradually increased -RRB- in order to obtain a continuous flow rate -LRB- 1 ml/min -RRB- and concentration of organic modifier -LRB- of 10 % -RRB- after mixing in the post-column counteracting gradient . This results in a constant flow rate and organic modifier concentration -LRB- after the 1:9 split -RRB- when entering the on-line PAD . The added value of this approach is that alterations in the chromatographic method can be made without much effect on the on-line PAD assay . First , the individual test compounds were analyzed . The test compounds were injected -LRB- in triplicate -RRB- in five different concentrations prepared by serial dilution of 100 μl of stock solution with 300 μl MeOH -LRB- 30 % v/v -RRB- . Typical superimposed chromatograms of menadione and L-ascorbic acid are shown in Fig. 4a and b , respectively . At high concentrations of L-ascorbic acid , two peaks were seen . This was the result of overloading the analytical column , as additional mass spectrometry data showed that both peaks were from L-ascorbic acid -LRB- data not shown -RRB- . The relative pro-oxidant or antioxidant response of every test compound in the PAD system used in gradient HPLC mode is shown in Table 1 . The sensitivities obtained for the test compounds are also depicted in Table 1 . When comparing the PAD system used in HPLC mode with the PAD system used in FIA mode and the off-line batch assays , it is seen that all three assay formats allow the detection of both pro-oxidants and antioxidants . The differences between the three assay formats probably result from different factors , such as the pre-incubation step applied in the off-line batch assay format and the peak broadening of compounds during the chromatographic separation . Another possible cause derives from minor pro-oxidant or antioxidant impurities in the test compounds . As these impurities would be separated from the test compounds in the on-line PAD system used in HPLC mode , they would only influence the results obtained in the off-line batch format and the on-line PAD system used in FIA mode . However , comparable results are still obtained -LRB- no more than threefold differences in measured pro-oxidant or antioxidant effects were obtained per mole of test compound -RRB- in the different experimental set-ups . Therefore , it can be concluded that the PAD system used in gradient HPLC mode can be easily applied to and is a sensitive method for the screening of individual compounds for their ROS-producing capacity and antioxidant potentials . Fig. 4a Superimposed PAD traces of menadione injected in different amounts into the PAD system in HPLC mode -LRB- 12.5 , 50 and 200 μM from bottom to top chromatogram , respectively -RRB- . b Superimposed PAD traces of ascorbic acid injected in different amounts into the PAD system in HPLC mode -LRB- 50 , 200 and 800 μM from top to bottom chromatogram , respectively -RRB- The PAD system used in HPLC mode was also applied to the detection of individual antioxidants and ROS-producing compounds in mixtures . Typical PAD traces of two different mixtures that were injected and separated on HPLC are shown in Fig. 5a and b , respectively . Figure 5a shows that all three ROS-producing compounds -- paraquat , menadione and duroquinone -- were individually identified as oxidant species . The compounds in the second mixture , which contained two antioxidants and two ROS-producing compounds , were individually identified as antioxidants -LRB- L-ascorbic acid and glutathione -RRB- and pro-oxidants -LRB- menadione and duroquinone -RRB- . Thus , mixtures in which ROS-producing compounds are present together with antioxidants could effectively be measured individually with the present PAD system in gradient HPLC mode . When analyzing such mixtures with traditional off-line batch assay formats , antioxidants can counteract the effects of pro-oxidants thereby reducing or even totally removing ability to detect the pro-oxidants . The relatively low resolutions obtained in the PAD assay directly reflect the resolutions resulting from the chromatographic part of the total system . Since this study was a proof-of-principle study , we did not perform a thorough optimization of the chromatographic part of the system . The resolutions that can be obtained with the PAD part of the system -LRB- used in FIA mode -RRB- are shown in Fig. 2 and reflect the performance of the assay . Connecting the PAD part to a chromatographic separation system with higher resolution will obviously result in higher resolution , which may be needed when screening real life samples . The present PAD system in HPLC mode therefore opens up new avenues to the efficient and rapid screening of complex mixtures for individual pro-oxidant and antioxidant components . Fig. 5a PAD trace for a mixture of three pro-oxidants injected into the PAD system in HPLC mode . Injected compounds are : paraquat -LRB- 0.12 mM ; 3.5 min -RRB- , menadion -LRB- 0.05 mM ; 17.5 min -RRB- , and duroquinone -LRB- 0.17 mM ; 18.5 min -RRB- . b PAD trace of a mixture of two pro-oxidants and two antioxidants injected into the PAD system in HPLC mode . Iinjected compounds are : ascorbic acid -LRB- 0.14 mM ; 5 min -RRB- , glutathione -LRB- 0.4 mM ; 8 min -RRB- , menadion -LRB- 0.05 mM ; 17.5 min -RRB- , and duroquinone -LRB- 0.17 mM ; 18.5 min -RRB- Conclusion This paper presents the development and validation of a HRS-based on-line post-column detection system for the detection of ROS-producing compounds as well as antioxidants in mixtures . Different parameters , such as substrate -LRB- 4-HPAA -RRB- and enzyme concentrations , reaction time , temperature , additives , and organic modifier concentrations were first optimized for the PAD system used in FIA mode . Several ROS-producing compounds as well as antioxidants were successfully measured with the optimized system . The intraday and interday variabilities of the PAD system used in FIA mode were determined and found to be lower than 5 % . Good sensitivities , at least comparable with similar off-line batch assay formats for individual compounds , were obtained . On-line coupling of the novel PAD system to gradient HPLC permitted the screening of individual compounds in mixtures for ROS-producing and antioxidant properties . It should be noted , however , that compounds that show fluorescence quenching or intrinsic fluorescence may interfere with the methodology . However , it may be possible to adjust the system so that another split directs some of the flow to a second on-line assay that measures these artifacts with a `` negative control PAD system '' in order to detect such interferences . This PAD system used in gradient HPLC mode is potentially of great value to drug discovery and toxicology and food research .

O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

antioxidant
ros
bioactivation
biochemical detection