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1. Determination of Serum Albumin
Serum albumin levels can be determined by standard electrophoresis, salt fractionation, or dye-binding techniques. Although electrophoretic analyses can provide precise, specific quantitative data, for this the technique requires an electrophoresis apparatus and densitometer or colorimeter and a separate determination of total protein. In contrast, the salt fractionation procedure is rather non-specific and suffers from lack of precision. On the other hand, specific dye-binding methods are rapid, involve few manipulations and specialized equipment, and do not require a separate determination of total protein. A widely used method of this type, based on bromcresol green (BCG) (12), is described here.
Principle
The addition of albumin to a solution of bromcresol green in a 0.075 M succinate buffer, pH 4.20, results in an increase in absorbance of 628 nm. The absorbance-concentration relationship is linear for samples containing up to 6 g/dl albumin. Bilirubin, moderate lipaemia, and salicylate do not interfere with the analysis. The use of a non-ionic surfactant (Brij-35) reduces the absorbance of the blank, prevents turbidity, and provides linearity. The results with this method agree very well with those obtained by electrophoresis and salt fractionation. The method is simple, it has excellent precision, and the reagents are stable.
Apparatus
Spectrophotometer or colorimeter equipped with 630 nm interference filter.
Reagents
Procedure
A sample of 25 µl of serum or working standard solutions are added to 5.0 ml of working dye solution. The solution is vigorously mixed and allowed to stand 10 minutes at 25° C. The absorbance is measured at 628 nm after adjusting the instrument to zero absorbance with the working dye solution. If a serum sample is extremely lipaemic, a serum blank is prepared by adding 25 µl of the sample to 5.0 ml of 0.075 M succinate buffer. Its absorbance with water as a reference is subtracted from the absorbance of the unknown. The albumin concentration of the serum is obtained from an absorbance-concentration plot, or, if the response of the instruments is linear, only a single standard solution (2.0 g/dl) is required and the serum albumin concentration is calculated in the usual manner. A typical standard curve is seen in figure 4.A.1 (see FIG. 4.A.1. The Absorbance-Concentration Relationships of the BCG Method).
2. Determination of Serum Prealbumin by Radial Immunodiffusion (RID)
Procedure as Described by Manufacturer:
Although the antibody for human prealbumin can be developed by standard techniques, commercial kits are available from Calbiochem-Behring Corp. (10399 North Torrey Pines Rd., La Jolla, CA 92037, USA) and may be more economical if a limited number of assays are anticipated. Since prealbumin is synthesized in the liver, acute and chronic liver disease will reduce serum concentrations independent of protein nutriture. Total fasting for more than 48 hours also will reduce serum levels by limiting the substrate available for hepatic protein synthesis. Hence, in the absence of liver disease and prolonged absolute fasting, prealbumin is a sensitive parameter for improvement in protein nutritional status.
Principle
A protein (antigen) solution is applied to a cylindrical well cut in a gel matrix containing a uniform concentration of monospecific antibodies. Antigen placed in the well diffuses radially, producing a precipitin ring. Precipitin rings can be read any time after overnight incubation, or endpoint. Results are quantitated by comparing the diameter of the precipitin ring produced by the sample to the precipitin rings produced by standards of known concentrations.
Equipment
Procedure
Plate preparation: Carefully remove the plate from the container. Open the plate by pressing the thumbs firmly on the centre of the lid while holding the lid at edges. Allow the plate to stand open at room temperature to permit evaporation of any moisture that may have condensed in the wells.
The user can choose, according to workload requirements, to make determinations based either on an overnight readout from a reference curve or on an endpoint readout (after 48 hours). The initial steps of the procedure are the same for either method.
Calibration
The assay range of M-Partigen(tm) prealbumin radial immunodiffusion plates is indicated on the label. If the sample concentration exceeds the upper limit of the assay range, the test should be repeated with appropriate dilutions of the specimen.
If the reference curve is not linear, or does not intercept the ordinate at 11 + 3.5 mm2 when the endpoint curve is plotted, the procedure should be repeated; errors in technique or product instability should be considered.
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
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
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
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 µg of retinol/dl. The factor 480 used for carotenes corresponds to a E 1%(1cm) of 2080 of this substance adjusted to yield directly µg of carotenes/dl. The respective calculations are as follows:
Retinol (µg/dl) = A° (328) - A' x 637
Carotenes (µg/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 µl 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
Reagents
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 µl 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 µl 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 µl chloroform-acetic anhydride reagent and 100 µl 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 µl 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 µl chloroform-acetic anhydride (1:1, v/v) reagent.
Then 100 µl 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
ß-carotene-blue colour at A450
Weigh exactly 50 mg freshly opened all bans ß-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 µg/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 µg/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)
µg carotene/ml. for carotene (C) where FC450 = (µg carotene/ml.) / A450
ß-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-ß-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 µg/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 µg/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: µg 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 µg/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 µg 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 µg/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 = (µg 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 (µg/dl) =A450 x FC450 x 150
Where FC450 is the constant determined in each laboratory and 150 accounts for dilution factors.
Vitamin A (µg/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
Haemoglobin concentration can be measured in venous or capillary blood by colorimetric determination of derivatives of hemoglobin such as cyanmethemoglobin, oxyhemoglobin, or acid hematin. Various automated methods exist that are based on some of these principles. The preferred method, as recommended by the International Committee of Standardization in Hematology (14), involves the conversion of ail hemoglobin derivatives except sulfhemoglobin to cyanmethemoglobin by dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Absorbance is then measured in a photoelectric colorimeter or spectrophotometer at a wavelength of 540 nm. Reference standards of cyanmethemoglobin that conform to the specifications of the ICSH are commercially available.
Comparable information to that obtained from hemoglobin determinations can be obtained by the measurement of the packed red-cell volume, a simple technique that only requires a microcentrifuge and capillary tubes. However, due to changes in the mean corpuscular hemoglobin concentration that occur in iron deficiency anaemia, changes in hemoglobin concentration are more marked than those in the packed cell volume.
Choice of methods for serum iron and total iron-binding capacity (TIBC) will depend on equipment and amount of specimen available. Most current methods consist of the colorimetric measurement of an ironchromogen complex and require a spectrophotometer. The TIBC is determined by adding excess iron to the serum sample and removing unbound iron by some absorbent such as magnesium carbonate. Percent saturation of the TIBC (transferrin) is then calculated. Careful handling of specimens with iron-free material is essential. The Iron Panel of the ICSH has recommended reference methods employing bathophenathroline sulfonate as the colour reagent (15-16). These methods have recently been reviewed in detail (17).
Recommended methods for the determination of FEP (18-19) require a fluorometer and relatively elaborate extraction stages. An alternate method determines Zn protoporphyrin directly from a blood smear using a special apparatus called a hematofluorometer (20). This method is simple, inexpensive, and well adapted for field work, but needs further validation. According to some authors (19), the instrument does not have the precision or accuracy to detect small changes in fluorescence that occur with mild iron deficiency.
Ferritin can be determined in a small amount of serum or plasma by radio-immunoassay (21), and there are several commercial kits that give satisfactory results. The need for radio-isotope facilities has been recently eliminated by the introduction of enzyme immunoassays that only require a spectrophotometer. EIA methods seem to give results comparable to those obtained by RIA. The relatively large variability of results that currently exists among different laboratories may be reduced in the future by the use of ferritin standards that are becoming available from the ICSH.
