Oxygen Radical Absorbance Capacitance Assays (ORAC) in Microplates

The antioxidant capabilities of foods, cosmetics, supplements and pharmaceutical agents have become of particular interest. This interest is the result of the evidence demonstrating the relationship of reactive oxygen/nitrogen species with aging and pathogenesis. [1,2]. Living organisms use of a fuel (ATP) whose production results in the formation of toxic compounds, requires that a balance be maintained between the oxidants and the antioxidants. ROS species are generally held in check by a combination of antioxidant enzymes, proteins, and antioxidants provided by the diet. A breakdown or reduction in anti-oxidant capability has been associated with a number of chronic diseases. Conceivably, the ingestion of foods or supplements that contain antioxidant activity could provide some protection towards this affect.





Figure 1. Illustration of Antioxidant activity determination expressed as the net area under the curve (AUC).


There are several different methods to measure total antioxidant capacity described in the literature (3-7). The oxygen radical absorbance capacity (ORAC) assay is the latest in a lineage of assays that attempt to measure the antioxidant capabilities of compounds and foods [7]. This assay has been automated [8] and over time converted to a microplate format [9]. The ORAC assay depends on the free radical damage to a fluorescent probe, such as fluorescein, to result in a downward change of fluorescent intensity [10]. The assumption of course is that the degree of change is indicative of the amount of radical damage. The presence of antioxidants results in an inhibition in the free radical damage to the fluorescent compound. This inhibition is observed as a preservation of the fluorescent signal. Reactions containing antioxidants and or blanks are run in parallel using equivalent amounts of a ROS generator and fluorescent probe. Because the reaction is driven to completion, one can quantitate the protection by calculating the area under the curve (AUC) from the experimental sample. After subtracting the AUC for the blank, the resultant difference would be the protection conferred by the antioxidant compound (Figure 1).

Comparison to a set of known standards allows one to calculate equivalents and compare results from different samples and experiments. Typically Trolox, (6-hydroxy-2,5,7,8-tetrametmethylchroman-2-carboxylic acid) a water soluble vitamin E analog, is used as the calibration standard and ORAC results are expressed as Trolox equivalents. The ORAC assay is unique in that because the assay is driven to completion the AUC calculation combines both the inhibition time as well as inhibition percentage of free radical damage by the antioxidant into a single quantity. Standardization of the assay with the use of a common calibrator in conjunction with an assay that can be performed easily on many different compounds, foods, and materials has allowed for an easy comparison of antioxidant capabilities of many different materials and the formation of a database [11].



Figure 2. Typical Antioxidant Standard Curve.

When net AUC are calculated from these kinetic curves and plotted against Trolox Concentration a linear relationship is observed (Figure 2). The standard curve can then be interpolated to quantitate unknown samples. The resultant determinations are expressed as Trolox equivalents. Several compounds known to have antioxidant properties were assayed using the ORAC assay as previously described. As depicted in Figure 3, all of these compounds show a significant linear concentration dependent antioxidant activity. The specificity of the assay is demonstrated by the lack of any response from Tris buffer.



Figure 3. Antioxidant Dose Response Curves. The ORAC of several different known antioxidant compounds, as well as Tris buffer were measured as described previously and their Net AUC plotted against their concentration.

The ORAC assay in our hands is very sensitive to slight temperature gradations. Despite extreme efforts, an edge effect was observed when the outside wells were used. Edge effects have been reported in any number of different types of microplate-based assays. Because were used the reader’s reagent injectors to initiate the assay, plate lids were not used. In these experiments, the tighter control between reading the wells and the initiation of the assay by the addition of AAPH was of greater value than the throughput. We found that filling the outer wells with 300 µL of water resulted in more consistent data from the interior wells. Besides avoiding the use of the outer wells, where most of the discrepancies were found, the filled wells served as a significant heat mass that eliminated any temperature fluctuations. In addition, we found that reading the plate from the bottom resulted in more consistent results.

References

1. Halliwell, B., Aruoma, O. (1991) DNA Damage by Oxygen Derived Species. Its Mechanisms and Measurement in Mammalian Systems. FEBS Lett. 281:9-19.
2. Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993) Oxidants, Antioxidants and the Degenerative Diseases of Aging. Proc. Natl. Acad. Sci. USA 90:7915-7922.
3. Benzie, IFF, Strain, JJ. (1996) The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 238:70-76.
4. Rice-Evans, C. and Miller, NJ. (1994) Total Antioxidant Status in Plasma and Body Fluids. Methods Enzymol. 234:279-293.
5. Wayner, DDM, Burton, Gw., Ingold, KU., and Locke, S. (1985) Quantitative Measurement of the Total, Peroxyl Radical-trapping Antioxidant Capacity of Human Blood Plasma by Controlled Peroxidation. FEBS Lett. 187:33-37.
6. Glazer, AN., (1990) Phycoerythrin Fluorescence-based Assay for Reactive Oxygen Species. Methods Enzymol 186:161-168.
7. Cao, GH., Alessio, HM.and Cutler, RG., (1993) Oxygen-radical Absorbance Capacity Assay for Antioxidants. Free Radical Biol. Med. 14:303-311.
8. Cao, G., Verdon, C., Wu, A., Wang, H., and Prior, R. (1995) Automated Assay of Oxygen Radical Absorbance Capacity with the COBRAS FARA II. Clin. Chem. 41:1738-1744.
9. Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J., and Prior, R. (2002) High-throughput Assay of Oxygen Radical Absorbance Capacity (ORAC) Using a Multichannel Liquid Handling System Coupled with a Microplate Fluorescence Reader in 96-Well Format. J. Agric. Food Chem. 50:4437-4444.
10. Ou, B., Hampsch-Woodill, and Prior, R. (2001) Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. J. Agric Food Chem. 49:4619-4626.
11. Wu, X., Gu, L., Holden, J., Haytowitz, D., Gebhardt, S., Beecher, G., and Prior, R. (2004) Development of a Database for Total Antioxidant Capacity in Foods: a Preliminary Study. J. Food Composition and Analysis. 17:407-422.
12. Cao, G., and Prior, R. (1999) Measurement of Oxygen Radical Absorbance Capacity in Biological samples. Oxidants and Antioxidants. Methods Enzymol. 299:50-62.

Epoch Contest Winner Announced!

In our blog How Low Can you Go? on September 17th we invited our readers to register to win the Epoch™ Multi-Volume Spectrophotometer System. The drawing was held on Friday, November 13th.

The Winner of the Epoch "Win Me" Contest, chosen at random from over 700 registrants from 69 countries, is Mr. Joël Tardivel-Lacombe from Institut National de la Santé et de la Recherche Médicale (INSERM), Luminy Campus in France.

Mr. Tardivel-Lacombe noted: "We have a lot of uses for the Epoch, in the UV and visible ranges, and in various volumes. In the lab we are using a BioTek ELx800 for apps in visible light in culture plates, a UV spectrophotometer for RNA, DNA and protein quantitation in cuvettes, and a NanoDrop forquantitation in small volumes. With the Epoch we’ll do all in the same machine! It’s a great improvement!"

BioTek’s Epoch™ Multi-Volume Spectrophotometer System consists of the monochromator-based Epoch Microplate Spectrophotometer controlled by Gen5™ software, and the unique Take3™ Multi-Volume Plate that measures up to sixteen 2 µL samples, two BioCells, or a standard cuvette. Laboratories are provided with a high value solution for multiple users, multiple assays, and preservation of precious nucleic acid and protein samples by taking measurements in 2 µL microspots, 6- to 384-well microplates, or cuvettes.

Thank you to everyone who registered in the contest and congratulations to our winner!

The Process of Building a Better Lab

After many years, BioTek has moved forward with plans to upgrade its application laboratory. While the original plan was to renovate the existing space, with the need for BioTek to expand its work space it was decided to build a new lab in newly renovated space. Yes, “A Brand New Lab!” While the saga is a bit long I feel that it is representative of many lab renovations as they move from the concept stage to fruition.

Location, location, location…
Designing a new lab space is very similar to designing space for home renovation and all in all quite fun. There are a number of guides on how to design a lab, but to get started, you really only need two pieces of information. (1) What function is the lab space intended to serve? In this instance, the application lab is a general purpose lab with tissue culture capability with a staff of approximately 5. (2) What space is available? The space indicated for the applications lab was approximately 23’ x 26’, which was partitioned into a general lab work area and a tissue culture room. Once the rooms were more or less sorted out now it was time to decide what we wanted for bench space. It’s a lab so we need lots of counters and storage. The current lab has an island in the center which I like a great deal so this type of setup would be incorporated into the new lab space. Island type counters, unlike peninsula allow access from all directions making movement around the lab easier. In addition to island bench space, a great deal of wall mounted cabinetry with counters would also be included. This should allow sufficient space for the plethora of equipment that already resides in the applications lab. Once the space considerations have been decided upon, its time to put pen to paper and layout the space. If you are like me, you better make it pencil, as there will be a few changes before you get things right. I prefer the hand drawn method of design, but the use of computer aided design will work just as well.

Scale, scale, scale…
Now that you have “planned” the design, now it is time to work with actual dimensions. If you have drawn everything to scale already things are pretty much already done when you complete the planning stage. Because the space intended for the applications lab was integrated with other departments, the overall space was always in a state of flux, depending on the whims of others. In my case the wall dimensions seemed to fluctuate daily, but over time the space seemed to reach some sort of steady state. In terms of the cabinetry, we decided to purchase lab cabinetry and counters. Unlike kitchen cabinets and counters, laboratory fixtures need to be chemical resistant. The old lab fixtures were surplus kitchen cabinets and countertops and were in process of delaminating. I contacted two different cabinet vendors and met with both. I explained what I wanted, provided scaled drawings and asked them for quotations. To their credit, both responded promptly with specific drawings and quotations. Neither was very close to what I explained or to the drawings that I had provided. After a series of phone conversations, e-mail exchanges and a second meetings, drawings that closely resembled what we were interested in eventually evolved. In regards to laboratory cabinets, there are really two choices for materials; steel or laminated composite. Steel last longer and is stronger, but is approximately twice the price. Laminate, while not as robust fit our budget better than steel. In the end, both quotes were very close to one another, but during the process I got a sense that one company had a better handle on lab design while the other specialized in office design. This made the choice pretty easy. Now we had to pick colors…

Stuff, stuff, stuff…
In addition to the cabinetry items such as safety equipment (i.e eyewash and safety shower), sinks, faucets etc have to be decided upon, along with their location. Large equipment purchases such as freezers (-80°C, -20°C) refrigerators, autoclaves, tissue culture incubators, biosafety cabinets, floor model shaking incubators, etc. all had to be purchased and places designated in the new lab space. These items can be purchased new and/or used. We used a combination of both to outfit the lab space. As the budget was not eligible for TARP funds, not everything could be purchased in the same physical year. Hopefully, we will get to buy some more presents for ourselves next year.

Change, change, change…
While all of the planning is taking place several things are happening outside one’s control that directly impact all of your well made plans. As I mentioned previously, others are involved in the overall design so wall positions and dimensions are still changing as a result of newly discovered code requirements, discrepancies in the original layout plans, the need for janitors closets, pre-existing plumbing design, builder’s whims, etc. With a 6 inch change here and a 6 inch change there, your carefully laid plans for equipment placement is soon a piece of fiction. The plan is soon obsolete and in need of being changed.

Decisions, decisions, decisions…
After the basics equipment has been chosen now it s time to decide water and electrical requirements. This is basically answering a number of questions: Where do I need power outlets? How high off the floor do they need to be? Are they 110V or 220V? Do they require a special outlet? The island cabinets require power that is dropped from the ceiling. The plumbing questions are a bit more basic. What size fitting do you need? Does it require a drain? Answering these questions of course requires that you know where everything is to be located, which as I explained previously can change. Fortunately I wasn’t asked about the color of the switch covers or outlets.

Time, time, time…
You have ordered the cabinetry along with the installation based on a predicted finish date supplied by the builders. You have mentally checked that piece of work off from your list of things to do only to find out that the completion date has been put off. This requires that you contact the cabinet manufacture to find out if you can push the delivery date back; only to find out that the cabinets have already been manufactured and that it will cost more to store them for a period of time. Meanwhile boxes of other purchased items are starting to pile up outside your office. Having learned your lesson, you pad the new delivery date by an extra week, only to find out even that date was optimistic and you need to contact the cabinet guys a second time. If you are fortunate vendors for other installed items are flexible in the installation date. As the days progress, boxes of parts and equipment begin to form a mountain around your office and your co-workers make polite inquiries as to when the mess of boxes will be removed.

Help, help, help
At this stage the installers are now busy at work. As one begins to see the design taking shape it becomes obvious that some things need to be changed. Much like working on your own car, the parts you have ordered don’t quite fit and there seem to be parts left over. In our situation, the base cabinets didn’t quite fill a wall. Our solution was to leave a gap in the center of the cabinet run that will make a nice spot for a trash bin. In addition we had an extra upper cabinet that was installed on another wall. As we were installing the center shelving for the island work areas it became obvious that some of the shelving was not necessary. The section we omitted should work nicely for the automation support work the apps group performs. The electrical drops to the island needed some customization as well. With the assistance of very good installers all of these issues were dealt with. After you think that everything has finally been dealt with and the lab is coming together you realize that a 36” freezer will just not fit through a door with a 34.5” opening. After a short period of panic regarding all of the other equipment that you have ordered, you realize that if you open the freezer door as you pass through the doorway that things will fit. On to the next crisis…

Move, move, move…
The day has finally arrived. The space renovation has been completed and you can now move into the new space. Only now do you realize exactly how much new stuff has been ordered, along with how much instrumentation you plan on moving from the old laboratory space. Time to purge some stuff. With good fortune there will be a facilities manager that will arrange for assistance to move much of the material. Regardless, plan on spending considerable time and energy (mental as well as physical) with this task. With the placement of instrumentation in the new space you find that carefully thought out planning only goes so far. Much of the layout design will work, but expect to have made a number of misjudgments. It will take some time to work out the best layout.

End, end, end…
The bottom line is that with some hard work and a large pile of money you too could have new laboratory space. In my case, a number of different people provided valuable assistance, without their help the new lab would not have happened. As with any renovation, a positive attitude and sense of humor go a long way toward making the process tolerable. In the end, the result was worth it.

MipTec 2009 Recap

MipTec, 2009 convened October 13-15 in Basel, Switzerland. Apart from the Swiss national soccer team securing a berth to the 2010 World Cup in South Africa, a highlight during the conference was certainly the talk given by Martin Mackay, President of Pfizer Global R&D: “R&D – As if our lives depended on it.” One of the key messages of his seminar was “choosing the right target for the right patient.” The right target is chosen by rigorous target validation. The explosion of genomics and microarray technologies in the ‘90’s ensured that there were copious amounts of putative drug targets associated with disease, but these were rarely properly validated. Some point to lack of validation as the causative agent for the decline in new chemical entities in this decade. Martin Mackay’s organization is collaborating extensively with academia to “choose the right targets” for their drug programs.

My mind goes back to an award lecture given by Leroy Hood at the 9th Annual SBS conference in Portland, Oregon titled “Systems Biology: Decifering Life.” This was 2003 and Hood had recently co-founded the Institute for Systems Biology (ISB). One of the most provocative statements he made in his lecture was that Pharma would never adopt systems biology approaches as they were too complex and would require too much of an investment before any financial returns were realized. This is why he set up ISB. Systems biology approaches and other rigorous methods for understanding diseases is largely the realm of academia or other publicly-funded organizations, where return on investment is typically gauged by the number of publications and their impact factors. Science typically wins out in the competition for funding.

I think Martin Mackay’s collaborations bode well for Pfizer’s future product pipeline.

ISSX 2009 North American Regional Meeting Recap

The continued importance of early ADME-Tox testing of lead compounds was the theme of the 16th North American Regional ISSX meeting. While much emphasis continued to be placed on testing for drug-drug interactions with cytochrome P450 enzymes, the most important family of primary metabolic enzymes, it was also pointed out that drug-drug interactions with transporter enzymes can also cause safety concerns with lead compounds. A large portion of a number of podium presentations concentrated on the importance of transporter enzymes in the distribution and elimination of compounds from the body. This was further emphasized by the FDA during their presentation on regulation of drug safety testing.

While LC/MS/MS is still an important readout for transporter enzyme assays, other detection methods suitable for microplate assays are coming to the fore which can use ubiquitous inexpensive detection instruments. An example of this is Solvo Biotechnology‘s P-gp PREDIVEZ Vesicular Transport Assay Kit, which uses fluorescent substrates to monitor activity and inhibition of this important efflux transporter enzyme.

BioTek plans to partner with reagent vendors such a Solvo, to demonstrate the automation and detection of these, and other ADME-Tox assay chemistries. This was demonstrated at the recent ISSX conference, where we presented two posters showing the automation and detection of luminescent primary and secondary metabolic enzyme assays.

What areas of ADME-Tox research do you feel will be important for drug safety testing? What throughput needs do you have to test the ADME-Tox properties of your lead compounds? How can BioTek help you to make this area of drug discovery easier, faster, and more reproducible?