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close this bookMethods for the Evaluation of the Impact of Food and Nutrition Programmes (UNU, 1984, 287 p.)
close this folder4. Measuring impact using laboratory methodologies
View the document(introduction...)
View the documentIntroduction
View the documentDevelopment of a primary nutritional deficiency
View the documentChoice of tissue for laboratory assessment
View the documentSelection of laboratory methodologies for nutritional impact evaluation
View the documentLaboratory methods for assessment of nutritional impact
View the documentAnnex A. Laboratory evaluation of protein nutriture
View the documentAnnex B. Laboratory evaluation of vitamin A nutriture
View the documentAnnex C. Suggested methods for hematology
View the documentReferences

Annex B. Laboratory evaluation of vitamin A nutriture

1. Spectrophotometric Method Based on UV Inactivation (1)

Principle

Vitamin A (retinol) is destroyed when exposed to ultraviolet light. After saponification with alcoholic KOH, retinol (and carotenoids*) is extracted by solvent partition using a mixture of xyiene-kerosene. The optical absorbance of the sample extract is read at 460 nm for the determination of total carotenoids and at 328 nm for the determination of retinol. The sample extract is then irradiated with ultraviolet light and its absorbance read again at 328 nm. The difference in optical absorbance at 328 nm before and after irradiation of the sample corresponds to the amount of retinol present. The concentration of carotenoids and retinol are calculated. based on their respective extinction coefficients in the solvent mixtures.

Equipment

  • A UV-VIS spectrophotometer with appropriate cuvettes. Recommended: When the amount of sample to be read is equal to or less than 1 ml, use a spectrophotometer with a microcell adaptor.
  • Vortex mixer
  • Centrifuge (preferably refrigerated)
  • Appropriate test tubes and glassware
  • Automatic pipettes (Eppendorf or Oxford type)
  • A weak ultraviolet light source. Recommended: Blacklight tubes, General Electric F 20 T 12/BLB, 20 watts or Eye FL 20 SBLB.

Since cleanliness is a critical factor in this type of analysis, it is recommended that all the glassware be treated as follows: After regular washing, glassware should be rinsed with a 50 per cent solution of nitric acid and rinsed again with sufficient distilled water to remove all traces of nitric acid. The microcells are first washed with a 1: 1 mixture of 3N HCI-ethanol, rinsed again with ethanol, and finally with the xylenekerosene mixture.

Reagents

  • Absolute ethanol
  • 11 N KOH: Prepared periodically from KOH pellets and stored under refrigeration, in polyethylene bottle.
  • Alcoholic KOH, IN: Prepared just before use by mixing 11 N KOH and absolute ethanol in a ratio of 1: 10. The leftover alcoholic KOH is discarded.
  • Xylene (certified ACS xylenes, Fisher Scientific Company). It must be additionally purified by distillation in the laboratory as follows: Distil at around 130-133 C using all-glass distillation equipment. Discard approximately 100 ml of initial and end fractions. The distilled xylene should read about 0.025 absorbance at 328 nm against water. Store in a dark bottle.
  • Kerosene (odorless kerosene, Fisher Scientific Company). It must be additionally purified in the laboratory as follows: Distil at around 193-198 C using all-glass distillation equipment. Discard respectively, as in the case of xylene, 100 ml of initial and end fractions. Purify further with norite and then filter by twice passing the kerosene-norite mixture through a double layer of Whatman No. 1 filter paper. The kerosene purified in this fashion should read less than 0.02 absorbance at 328 nm against water. Store in dark bottle.
  • Kerosene/xylene mixture. Mix equal volumes of purified kerosene and xylene just before use.

Standard

In this method, the introduction of a standard serves only to check the assay conditions, and its absorbance is not used in the calculation of the retinol concentration.

For the purpose specified, the USP reference standard for vitamin A from USPC Inc., Rockville, MD, USA, can be used. This is a solution of retinol acetate in cottonseed oil. Several procedures to prepare adequate standard solutions using this substance have given excellent results. One is outlined below. Weigh out approximately 30 mg of the oily UPS reference standard solution of vitamin A, which contains approximately 34.4 mg retinyl acetate/g, and dissolve in 100 ml ethanol. The solubility of the oil in ethanol does not permit more concentrated solutions. This solution can be used as a stock if kept in a dark bottle and refrigerated. If a calibration curve is desired, prepare suitable dilutions of the stock standard in ethanol. Carefully determine the absorbance of these solutions at 328 nm before (A0) and after (A1) irradiation with ultraviolet light.

Determine the time required for A, to reach a plateau under the assay conditions. Calculate net absorbency values (A0 - A1) of retinyl acetate solutions for each dilution used under conditions of optimal bleaching. Under proper conditions of irradiation, A, should be 3 per cent or less of A0. Plot the values with A0 - A1 on the y axis and relative vitamin A concentration on the x axis curve. Determine the method-specific absorbency factor as a function of the retinyl acetate concentration. For the latter, an E 1%(1cm) value at 328 nm of 1565 for retinyl acetate in ethanol is used. To obtain the concentration of the retinyl acetate solution in terms of retinol, an E 1%(1cm) value of 1795 in ethanol is used. This value corrects for the 3 per cent decrease in the E 1%(1cm) value of retinyl esters in ethanol.

Procedure

  1. Place in appropriate test tubes duplicated aliquots of 200 l of distilled water for blanks, standard solution (if desired), a control serum and unknown samples. For a small sample size like this. slender test tubes such as 6 x 50 mm or 10 x 75 mm are recommended. If starting with frozen aliquots of sample, thaw gently in an ice water bath at approximately 10-15 C.
  2. Add 200 l of alcoholic KOH to all tubes (including blanks) and mix well on the vortex mixed for 10-20 seconds. Stopper all tubes.
  3. Place the tubes in a water bath at approximately 55-60 C for 20 minutes. In our experience, temperatures even slightly above 60 C. may result in turbidity of the extracts.
  4. Prepare a 1: 1 mixture of xylene-kerosene while the samples are saponifying in the water bath.
  5. After 20 minutes, cool samples to room temperature and add 200 l of the xylene kerosene mixture. Extract retinol by vigorous mixing of each tube on the vortex for at least 30 seconds.
  6. Centrifuge for 5 minutes at 600-1000 xg.
  7. Withdraw the xylene-kerosene supernatant by means of a constriction micropipette connected to a rubber tube (for mouth sucking) or to a Pasteurpipette and place this sample extract in the spectrophotometer cuvettes. Read the optical absorbance at 328 nm for retinol and 460 nm for total carotenoids. Avoid contamination of the upper organic layer with aqueous phase, since re-mixing may cause turbidity.
  8. Transfer the sample extract from the cuvette to glass tubes (10 x 75 mm) for irradiation. Irradiate all the samples and blanks for 35 minutes using an ultraviolet irradiation source ("black light unit"). The time required for extensive bleaching may vary, depending on the light source used and other assay conditions. In one test laboratory, 60-90 minutes of irradiation were needed. Transfer the irradiated sample extract to the cuvettes and again read their optical absorbance at 328 nm.

Calculations

The calculations for retinol and carotene are based on their respective extinction coefficients (E 1%(1cm). The factor 637 is used to calculate the retinol concentration and it corresponds to a E 1%(1cm) of 1,570 adjusted to yield directly g of retinol/dl. The factor 480 used for carotenes corresponds to a E 1%(1cm) of 2080 of this substance adjusted to yield directly g of carotenes/dl. The respective calculations are as follows:

Retinol (g/dl) = A (328) - A' x 637
Carotenes (g/dl) = A (460) x 480 where A = initial optical absorbance reading
A' = optical absorbance after ultraviolet irradiation

Effect of Sample Storage

If care is taken to minimize the air/serum interface (storage under nitrogen is strongly recommended) and exposure to light, samples may be stored at -20 C for several months without affecting the vitamin A levels. Retinol is more stable on storage than carotenes are.

Variations and Modifications

The size of the sample can be changed as desired from 50-200 l to larger volumes up to 1.5 or 2.0 ml, depending on sample volume availability. A large sample volume may facilitate the analytical procedure by avoiding the use of specialized glassware, micro-cell adaptors in the spectrophotometer, and the meticulous and sometimes difficult handling of small amounts of sample extracts.

Although the ratio of sample volume to the volume of alcoholic KOH added for saponification must always be 1:1, the ration of sample size to the volume of solvent added for the extraction procedure can be modified. Instead of using a 1:1 ratio, larger volumes of solvent (xylene-kerosene) can be used. This modification may facilitate the extraction procedure and will provide a larger volume of sample extracts that would be easier to handle. Keep in mind, however, that the more solvent added the more diluted the vitamin A content of the sample, and the lower the optical absorbance readings. This may be a critical factor to consider when dealing with samples with low vitamin A levels.

If the proportions of sample size to the volume of solvent added is changed from a 1:1 ratio, make sure that proper correction is made for the dilution of the sample when doing the calculations.

Because of its high boiling point, the xylene-kerosene mixture is the preferred solvent. Only kerosene with an initial O.D. below 2 is suitable. However, laboratory workers can use other organic solvents, such as purified jet fuel (Turbo-fuel A-1), which is available at most major airports. About 300 ml of the solvent are distilled in 15 ml fractions. Fractions with an O.D. < 0.5 at 328 nm are pooled for use. Appropriate E 1%(1cm) for retinol and carotenes must be selected for the specific solvent used. The volatility of cyclohexane, otherwise an excellent alternative, reduces its utility. This constraint is critical, especially when using small amounts of sample.

 

2. Colorimetric Method Based on Carr-Price Reactions Using TFA (1)

Principle

The proteins of plasma or serum are precipitated with ethanol and the vitamin A and carotene are extracted into hexane (or petroleum ether). The carotene concentration is determined by measuring the absorption of the extract at 450 nm (A450). Following evaporation of the solvent, vitamin A dissolved in chloroform is determined by reading, at two time points the intensity of blue colour developed after addition of trifluoracetic acid-chloroform reagent. A correction is made for the concentration of carotene, since carotene contributes to the intensity of blue colour when present in high amounts. When serum levels of vitamin A are low in the presence of carotene in high amounts. falsely low vitamin A values can be avoided by first removing carotenoids by chromatography on alumina columns.

Apparatus

  • Colorimeter or spectrophotometer, with appropriate cuvettes; adapted to take masked microcuvettes for the microprocedure
  • Vortex mixer
  • Glass stoppered test tubes
  • Pipettes, 0.1, 2.0, 3.0 ml (or 0.01, 0.05, 0.075 and 1.0 ml for microprocedure)
  • Centrifuge
  • Water bath
  • Nitrogen tank (or other inert gas)
  • Volumetric flasks, 10 ml, 100 ml.

Reagents

  • Ethanol, 95 per cent or absolute
  • Hexane (or Petroleum ether, BP/38-57 C)
  • Chloroform, reagent grade
  • Acetic anhydride, reagent grade
  • Trifluoroacetic acid, reagent grade (trichloroacetic acid can substitute)
  • Chromagen prepared just prior to use: 50 per cent TFA in chloroform (v/v) Vitamin A standard, USP Reference capsule of retinyl acetate in oil, or purified crystalline retinol or retinyl acetate
  • carotene standard, ail trans. Use only freshly opened ampules that have had the carotene sealed under nitrogen. Carotene is highly unstable and even unopened standards sometimes have deteriorated.

Macroprocedure

All procedures should be carried out in dim light and caution exercised to avoid excessive exposure to oxidation.

Duplicate 2 ml aliquots of serum or plasma are pipetted into glass-stoppered test tubes. An equal volume (2 ml) of ethanol is added dropwise with mixing to give a 50 per cent solution (v/v). At this concentration the proteinretinol blond is disrupted and the free retinol and retinyl esters are extracted by addition of 3 ml hexane (or petroleum ether). The tubes are stoppered and contents are mixed vigorously by mechanical mixer for 2 minutes, then centrifuged 5-10 minutes at 600-1000 xg to obtain a clean separation of phases. Then 2 ml of the upper hexane (or petroleum ether) extract is pipetted into cuvettes and the cuvettes capped. Absorbance at 450 nm due to carotenoids is read against a hexane (or petroleum ether) blank (A450).

After determining A450, the cuvettes are removed and the hexane (or petroleum ether) evaporated just to dryness under a stream of nitrogen in a 40-60 C. water bath in dim light. If evaporation cannot be carried out in the cuvettes, transfer to another tube with rinsing and proceed. Just at the point of dryness, the residue is immediately redissolved and dehydrated in 0.1 ml of a mixture of chloroform acetic ahnydride (1 :1 v/v). The cuvettes or tubes should be capped to minimize evaporation and protected from light.

The spectrophotometer at 620 nm is set at zero absorbance with a blank consisting of 0.1 ml chloroform-acetic anhydride mixture and 1.0 ml TFA-chloroform chromagen reagent.

The cuvette containing the sample is placed in the spectrophotometer and 1.0 ml TFA chromagen reagent added to the cuvette from a rapid delivery pipette. Alternatively, the reagent can be added to the extract in a separate tube and rapidly transferred to the cuvette. These steps must be carried out rapidly and with care since the blue colour fades quickly and the chromagen reagent is highly corrosive. Record the absorbance reading (A620) at exactly 15 seconds (t15) and 30 seconds (t30) after addition of the reagent (A620).

Microprocedure

The microprocedure is essentially the same as the macroprocedure except adapted to a smaller scale. Accurate results are dependent upon great care in pipetting and transfering small volumes and minimizing possible losses from evaporation, oxidation, and light exposure.

A minimum of 50 Ill and preferably 100-200 l serum or plasma are pipetted into 6 x 50 mm glass stoppered test tubes. An equal volume of ethanol is added with mixing followed by 1.5 volumes hexane or petroleum ether (40-60 BP) (1 :1: 1.5 v/v/v ratio serum-alcohol-solvent, respectively).

The tubes are immediately stoppered and vortexed for 2 minutes, then centrifuged 5-10 minutes at 600-1000 xg to achieve a clean phase separation. 100 l hexane (petroleum ether) extract is transferred to a microcuvette by means of a micropipette and the absorbance due to carotenoids at 450 nm read against a hexane (or petroleum) blank.

The spectrocolorimeter, or preferably a spectrophotometer of quality equivalent to a Beckman DU, is set at 620 nm and then zeroed against a reagent blank containing 10 l chloroform-acetic anhydride reagent and 100 l freshly prepared TFA-chloroform chromagen reagent.

The sample is then transferred from the microcuvette to a clean 6 x 50 mm test tube, cuvette rinsed once with 50 l hexane (petroleum ether), and the rinsing added to the sample in test tube. The extract is then evaporated just to dryness under a stream of nitrogen in a water bath in dim light.

The residue is redissolved in 10 l chloroform-acetic anhydride (1:1, v/v) reagent.

Then 100 l TFA-chloroform chromagen reagent is rapidly added with vigorous mixing and the solution rapidly transferred to the microcuvette by means of a microtransfer pipette.

A reading at A620 is obtained against a TFA reagent blank at exactly 15 sec. (t15) and 30 sec. (t30) after addition of the chromagen. Careful timing is essential since the colour fades rapidly.

Preparation of Standard Curves

carotene-blue colour at A450

Weigh exactly 50 mg freshly opened all bans carotene standard and dissolve in a few millilitres of chloroform. Bring to exactly 100 ml in a volumetric flask with hexane (or petroleum ether). Prepare just prior to use in establishing the calibration curve since the solution deteriorates on storage. This is the stock carotene solution containing 0.5 mg/ml. Protect from light.

An intermediate standard containing 5 g/ml is prepared by diluting 1 ml stock carotene solution to 100 ml in a volumetric flask with hexane (or petroleum ether). This solution is stable only for a few hours and should be made just prior to use.

Working standards are prepared from the intermediate standard by diluting with hexane (or petroleum ether) in each of four 10 ml volumetric flasks 1, 2, 4, and 8 ml of intermediate standard solution. This results in solutions containing 0.5, 1.0, 2.0, and 4.0 g/ml of ,13-carotene, respectively.

Fill the cuvette with the carotene working standards and read A450 against a hexane (or petroleum ether) blank. Plot a standard curve and from it determine the factor (F)

g carotene/ml. for carotene (C) where FC450 = (g carotene/ml.) / A450

 

carotene-blue colour at A620

Carotenoids react with the TFA-chloroform to add to the blue colour at A620. Therefore, it is necessary to run a chromagen-carotene standard curve in order to calculate a correction factor in obtaining vitamin A values. This correction is not necessary if serum (plasma) contains carotene in concentration under 50 g/dl as the contribution to the blue color in this concentration range is negligible.

Carotene standards in chloroform are prepared to contain 4.0, 8.0, and 10.0 g/ml. Aliquots of 0.1 ml are pipetted into cuvettes. To this are added rapidly 1.0 ml TFA-chloroform chromagen with vigorous mixing. Absorbance at 620 mm is read at 15 sec. (t15) and 30 (t30) sec. exactly as described previously. The t15 and t30 values are plotted on rectangular coordinate graph paper with the ordinate containing A620 values and the abscissa time after addition of chromagen. Extrapolate to to with a ruler to determine the A620 at to. The to absorbance value, thus, can be determined by the formula At0 = At5 + (At5 - At30). Determine a factor (FC620) where: g carotene/ml FC620= A

A620 The carotene correction factor for vitamin A at A620 is FC5620 C in which the factor 2 derives from the difference in the dilution of the carotenoids and vitamin A in their respective assays.

 

Vitamin A

Retinyl acetate or retinol can be used as standards for preparation of reference curves, since both have identical blue color characteristics in the analytic procedure after making appropriate molecular weight adjustments to convert retinyl acetate (MW = 328) to retinol (MW = 286) equivalents (i.e., when the acetate is used 286/328 = 0.872). The USP reference capsule of retinyl acetate in oil is suitable. It is said to contain 34.4 mg all bans retinyl acetate/gin solution, but it is necessary to check the concentration of dilutions by spectrophotometry. Standards should be kept refrigerated and protected from light. They should not be used beyond storage of two days without redetermining the concentration spectrophotometrically.

A stock vitamin A standard containing approximately 50/60 g/ml is prepared by carefully weighing an appropriate amount of the vitamin A standard and diluting with hexane in a volumetric flask. The exact concentration in g retinol/ml is obtained by determining the absorbance at 325 nm and using the retinol extinction coefficient, E 1%(1cm) = 1,850.

Working standards are prepared in 10 ml volumetric flasks by diluting the appropriate volume of the stock solution with hexane (or petroleum ether) to obtain concentrations in the range of 6, 12, 24, 36, and 60 g/ml.

Then 0.1 ml of each working standard is pipetted into cuvettes for reaction with 1.0 ml TFA-chloroform chromagen exactly as previously described, reading A620 at 15 sec (t15) and 30 sec (t30).

The A620 values for t15 and t30 are plotted on a graph where the ordinate is the A620 values and the abscissa the time after addition of chromagen. Using a ruler, extrapolate to to to obtain A620 for each working standard. This is the theoretical time of maximum colour intensity obtainable, since the decay in blue colour is linear at least up to 30 seconds after chromagen additions.

Plot a standard curve from the A620 values at to on ordinary rectangular coordinate paper where the ordinate is the A620 value and the abscissa the number of micrograms of vitamin A per tube. From the curve calculate a factor (FA620) where:

FA620 = (g vit A/tube) / A620

Calculations

Based on the procedure outlined using 2 ml serum (plasma) extracted into 3 MP solvent, serum values are calculated by the following formula:

Total carotenoids (g/dl) =A450 x FC450 x 150

Where FC450 is the constant determined in each laboratory and 150 accounts for dilution factors.

Vitamin A (g/dl) = (A620 - 2A450 x FC620) x FA62o x 75 FC620

If the microprocedure is used, appropriate modifications in the calculations must be made for the volume of serum actually used.

Storage Precautions

Following collection of blood, serum or plasma should be separated within 24 hours. Preferably, the analysis will be carried out immediately. If this is not possible, serum or plasma should be frozen at -20 C until analysed. The samples should be placed in tubes that allow a minimum air head space, tightly stoppered and protected from light. Stability is increased by flushing the tubes with nitrogen or other inert gas prior to a tight seal. Storage without thawing results in little or no loss up to about one month, with approximately a 10% loss over a four-month storage period. Storage in a freezer has been reported not to affect values when TFA chromagen is used. Freezing may induce spurious high values when antimony bichloride is used. Each laboratory should determine the stability of standardized samples under its own storage conditions.

Other precautions

  1. Maximum blue colour intensity develops at a TFA concentration of at least 40% in the final mixture. Adjustments that are made in reagent or sample volume must be such that it maintains at least a 40% final TFA concentration.
  2. The decay rate of blue colour may vary for standards and unfractionated serum (plasma) extracts, but in both cases the decay is linear at least up to 30 seconds. Greatest accuracy is achieved by extrapolation of absorbance readings at t15 and t30 to to, where theoretically the maximum colour intensity is achieved. Standard curves should also be based on the to readings.
  3. It is necessary to periodically check the concentration of vitamin A in the reference standards by spectrophotometry.
  4. The TFA reagent is highly corrosive and maximum caution should be used to avoid contact with skin or mucous membranes. Never pipette by mouth; similar caution obtains for chloroform.
  5. Vitamin A reading below 20-25 g/dl by the TFA procedure using a correction factor for carotenoid interference may be incorrect. For samples with vitamin A values in this range, preliminary removal of carotenoid contaminants should be done on approximately 6 per cent water reactivated alumina columns. The hexane extract of plasma is placed on the column, carotenoid contaminants eluted by addition of 10 ml hexane retinol, and retinyl esters are then eluted from the column by addition of 10 ml solution of 50 per cent ethyl ether in hexane (v/v). The TFA procedure is carried out as described above.
  6. Slight haemolysis does not interfere with the reaction (13) and there is no significant change on storage provided samples are not repeatedly thawed and refrozen. These points should be checked in each laboratory under actual conditions.