What is the normal reference range for TAS? The normal reference range is quoted as mmol/l

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1 What is the normal reference range for TAS? The normal reference range is quoted as mmol/l How sensitive is the TAS assay? The sensitivity of the test is dependent on the actual antioxidants being measured, since the test gives an overall indication of antioxidant protection in a given sample. However we generally define the lower limit of measurement as 0.1mmol/l. Is TAS run routinely in any labs? TAS is performed routinely in a number of countries, namely, Korea, India, Switzerland and Austria. Can citrated tubes be used for sample collection for TAS? Citrated tubes are not considered suitable. Citrate is not simply an anticoagulant, but has complex effects on blood biochemistry via the citric acid cycle, some of which involves oxidative steps. Since the TAS assay measures overall antioxidant status this is thought to include interactions between antioxidants, if an anticoagulant is used it should be as simple as possible, so as to avoid potential effects on the antioxidant system. Does EDTA affect the TAS assay? EDTA should not affect the TAS assay when it is used in appropriate concentrations. If using EDTA in anticoagulant tubes, these tubes MUST be filled to the line advised on the side of the tube. Failure to do this will lead to variation in the EDTA concentration of the sample i.e. do not put 1ml of blood into a tube meant for 10ml. The best type of EDTA tube to use is a glass tube containing K 3+ EDTA, although use serum or lithium heparinised plasma for TAS measurement wherever possible. Can TAS samples be read on a microtitre plate reader? We do not recommend this, mainly because the timing of the assay is very critical. Even if the customer is using a multichannel pipette to dispense the samples, there will be a delay between dispensing between the rows of the microtitre plate. This would then necessitate the same delay to be incorporated into the taking of the O.D. readings of those same wells (which is not possible). Since the reaction is not stopped, nor is it at endpoint at the read time, this is liable to lead to inaccuracies in TAS measurement Does Alcohol interfere with the TAS assay? Alcohol does not interfere with the TAS assay and there should be no antioxidant activity in the alcohol itself. Alcohol is the most suitable solvent for substances, which are not soluble in water, as it should not affect the TAS level of substances tested. Other solvents can be used but the solvent must be measured itself, so that any inherent activity can be allowed for

2 Does ethanol interfere with the TAS assay? There should not be any interference from 100% ethanol used to prepare samples. To be entirely certain of no contribution from any solvent, the solvent in question can be run as a separate sample, and any value subtracted from that of the sample value. Can TAS measure oily substances? These samples would not be suitable by themselves for TAS measurement, as they would not mix fully with the reagents. However these samples may be mixed with alcohol at a known dilution and assayed. Can turbid samples be assayed? Turbid samples are unsuitable for the TAS assay. What about using TAS in tea? Regarding tea measurement, we have found levels vary dramatically from one type of tea to another, and again it is feasible that this level would be detected. Some teas and infusions have very little antioxidant activity. In addition, antioxidant activity will vary with the time allowed for infusion. I generally allow 5 minutes in boiling water. Some of our everyday teas everyday teas are generally prepared at approx. 5g/l. We would generally expect to see results in the region of 10-20mmol/l. How should wine be analysed? As a general guideline white wine samples are assayed without dilution, whereas red wines require dilution, usually in the range of 1/10 to 1/20. Ideally where dilution of samples is necessary, samples should be diluted so as to produce values in the center of the assay range (approx mmol/l). The final result is then that value multiplied by the dilution factor. When red wine is oxidizing in the barrel, would you expect the TAS to go up or down? During the maturation process of wine, it would be expected that the TAS level would go down. The oxidative process itself would decrease the antioxidant content but also as the wine matures, the phenolics polymerize and fall to the bottom of the bottle or vat. Since these are responsible for a proportion of the antioxidant content TAS levels would be expected to decrease. Can TAS be measured in urine and saliva samples? Yes, although the validity of levels would have to be assessed, for example vit c would be rapidly excreted in the urine if excess were to be consumed. Also in saliva, possible contamination from whatever has been recently consumed could occur. Urine samples would very likely require dilution. We have not established normal ranges for these analytes.

3 Can TAS be run in milk? Currently there is no procedure written and we have no data on whether this will work successfully or not. The milk should be run both neat and in a series of dilutions to obtain a result in the most linear section of the curve, that is between 1 and 2 mmol/l. If the results are not reproducible then the sample will necessitate some preparation. The milk can be spun for 10 minutes at 5000 rpm to remove the solids in it. The supernatant can then be run neat and in a series of dilutions as done before. Again the desired results are between 1 and 2 mmol/l A protein analysis should be run on the samples before they are used, if the results are to be expressed related to it. Deproteinisation of samples will cause the results to be distorted as this is traditionally performed with strong oxidants. Can TAS be used to measure antioxidant levels in cell cultures? Although we have no data on this measurement being made, it is entirely possible. The following protocol is merely a suggestion. 1. Subculture the cells not later than 3 days prior to analysis. It is preferred that cells are harvested when they have just reached confluence. 2. Carefully remove medium from the cells and, if required, retain for analysis. If the medium is not to be analysed immediately, freezing is recommended (preferably at 70 c, or in liquid nitrogen). 3. Gently rinse the cells x 2 with 0.9% NaCl solution or PBS. 4. Draw off all the liquid from the flask with a glass pipette. Add a small volume of 0.9% NaCl or PBS (0.5ml 1.0ml for a T25 Flask). Using a cell scraper, scrape all the cells off the growing surface of the flask. 5. Draw off the cells and place in a centrifuge tube. Rinse the flask with a small volume of 0.9% NaCl or PBS to ensure that all cells have been harvested. 6. Spin the cells at low speed (1000rpm) to ensure that they do not lyse and remove the supernatant. 7. Gently resuspend in 0.9% NaCl or PBS, spin once more and remove the supernatant. 8. If cells are not to be analysed immediately they should be stored frozen (preferably at 70 c, or in liquid nitrogen). 9. Prior to analysis, resuspend in a small volume of 0.9% NaCl or PBS and sonicate for a few seconds, keeping the cell preparation on ice. 10. Perform a protein determination and use the cell preparation at a concentration, which gives results within the linear range of the assay. 11. Express cell results as activity per mg/ug/ng protein as appropriate, and media results as activity per m/l analyser Can TAS be used to measure antioxidant levels in plant extracts? Yes, the following procedure is preferred.

4 The material should be frozen in liquid nitrogen, followed by freeze-drying and grinding to a fine powder. Extracts can then be prepared by adding 70% methanol (30ml to 0.5g powder) and boiling for 15 minutes. Remove the methanol by rotary evaporation and adjust the sample volume with deionised water. Centrifuge for 5 min at 5000g. If the above facilities are not available, the material should be chopped finely and homogenized fully in a 70% methanol solution. The preparation should then be spun to remove any solid material. Methanol and ethanol do not affect the assay system, however if other solvents are to be used, a sample of this solvent should be run in the TAS assay so that any inherent antioxidant activity can be accounted for. Both preparations should be diluted with deionised water to provide a TAS value within the assay range, although to avoid excessive dilution, dilution to provide a reading of mmol/l is preferred. Depending on the extraction procedure used, results may be expressed per Τg wet weight or per Τg dry weight. Can TAS measure levels of antioxidants in tissue homogenates? Several customers have used TAS for measurement of antioxidant content of tissue homogenates. However, due to interference by haemoglobin, some tissues, in particular those which would have a high level of blood present, may not be entirely suitable for TAS analysis. There is known interference to the TAS assay from haemoglobin. The assay was originally intended for use in serum/plasma, but may also be used in beverages and food, where a suitable preparation can be made. When assaying tissue homogenates, it may be advisable to perform a protein assay on the preparation, and also to assess the haemoglobin content of the homogenate /supernatant so as to take into account the contribution to TAS from haemoglobin. The actual weight per volume of tissue is likely to vary from one tissue type to another. Whatever the preparation you should aim for values in the middle of the TAS range. This is achievable by dilution e.g. 1-2mmol/l, and then multiplying up by the dilution factor. Tissue should be washed in saline solution prior to homogenization, in order to remove surface blood contamination. Samples may be homogenized in buffer. Assay conditions (temp. time etc) must not be altered, as this will affect the result How should fruit extracts be prepared for TAS analysis? Fruit extracts may be prepared by either, Squeezing the fruit, if it suitable to do so (e.g. citrus fruit) or Use of a juice extractor Or For firmer fruit (e.g. berries) Weigh the fruit and homogenize on ice 2x15 second bursts at 9500rpm. Centrifuge at 2500rpm and remove supernatant for assay. If the fruit does not contain large quantities of juice, the preparation may be brought to a known volume with deionised water prior to homogenization. In this instance, any dilution factor should be taken into account.

5 If the supernatant requires dilution, dilute the extract so as to give a TAS value approximately in the middle of the range, i.e mmol/l The overall TAS value is then obtained by multiplying by the dilution factor. For a series of samples of the same nature, it is preferable that the dilutions used remain the same, unless the TAS values of any individual samples exceed the overall assay range. Results are usually expressed as mmol/l, however it may be useful in some instances to refer to TAS in terms of weight/vol. What antioxidants does TAS measure? The major antioxidants included in the TAS assay are ascorbate, protein thiols, bilirubin, urate and Ι - tocopherol. These chain breaking antioxidants are thought to act in the above sequence to counteract the effects of oxidative stress. Preventative antioxidants present in the plasma include caeruloplasmin and transferrin. These antioxidants contribute to the TAS result due to their ability to sequester iron. Together these antioxidants account for approximately 90% of the TAS value. The remaining 10% of the plasma TAS value is thought to include cysteine, glutathione, ϑ carotene, dihydrolipoate and ubiquinone. The assay will, therefore detect those antioxidants present which will function as reductants and hydrogen donors. What is Drabkin s solution? Drabkin s consists of sodium bicarbonate, potassium ferricyanide and potassium cyanide (it is the main chemical in our haemoglobin kit). We normally supply it in powder format, to be reconstituted in 1000ml deionised water, however when used with the Ransel kit for GPx detection, it is used at double strength (i.e. prepared in 500ml deionised water) Can Ransel be used on RBCs? Yes, using the exactly the same protocol as for whole blood. The whole blood should firstly be centrifuged and the plasma removed. Since the test results are best expressed as U/g Hb, a haemoglobin test should be performed on whole blood when analyzing for whole blood, or on red blood cells when using a RBC sample. We recommend Ransel be used for the determination of selenium dependent GPx in whole blood or RBCs. When plasma is used as a sample total GPx is measured, not just the selenium dependent form. Neat plasma samples are used in this case. Can Ransel, Ransod, and GR be used in tissue samples? Although we advise the use of Ransel, Ransod, and GR in blood, and have no direct experience ourselves of their measurement in tissues, it should be conceivable that these products could be used for measurement in tissues. However, it should be pointed out that in the case of Ransel, if erythrocytes are not used as the sample the assay will no longer be specific for selenium form of GPx, but will measure total levels. In RBCs, 99% of the GPx present is selenium dependent and so the assay relates in these circumstances to Se-Gpx, however the composition of the GPX isoenzymes will vary

6 drastically from one type of tissue to another. The Ransod and GR assays should not be subject to this problem. However, it will be necessary to prepare a homogenate of tissue for assay and the results may be best expressed in terms of activity per mg protein. How many tests can you get from each kit? Kit Prod. No. Manual Auto TAS NX RANSOD SD RANSEL RS macro semi micro RS macro semi micro RS macro semi micro The number of assays per kit is determined by dividing the volume of reagent provided (per bottle) by the volume required in the assay, and then multiplying by the number of bottles of reagent per kit. Where two reagents are provided, the one providing the least number of assays is the one from which the total number of assays is calculated. Automated test numbers are based on Cobas Fara requirements.