Microdetermination of Phosphorus P. S.
CHEN, JR., T. y. TORIBARA, and HUBER WARNER
Division of Pharmacology, Department of Radiation Biology, School of Medicine and Dentistry, University of Rochester, Rochester, N. Y.
The ascorbic acid method of Ammon and Hinsberg, modified by Lowry and associates, has been applied to of phosphorus in whole blood, the determination plasma, serum, and urine. A sensitivity about eight times that of the aminonaphtholsulfonic acid method permits the use of much smaller samples for measurement in conventional cells (as little as 0.15 7 of phosphorus can be determined in ordinary 3-ml. cuvettes.) A comparison with an accepted procedure on a number of samples showed that the ascorbic acid method gave essentially the same results.
Figure 2.
A
method for phosphorus sufficiently sensitive to dispense with microtechniques and special glassware and apparatus. This is true in cases where only small samples are available, or where a number of different analyses must be made on a limited amount of specimen. The procedure presented in this paper fills such a need. Most sensitive methods for determining phosphorus in biological materials are based on the color formed by the reduction of a phosphomolybdate complex. Probably the most widely used method is that of Fiske and Subbarow (3), in which the reduction is carried out with sulfite and aminonaphtholsulfonic acid.
NEED has existed for
a
Stabilities of solutions
Colors developed from reagents standing for indicated number of days Upper. Ascorbic acid kept at 4° C. Middle. Reagent C kept at 4° C. Lower. Reagent C kept at 25° C.
and Subbarow method (3). The procedure of Lowry and associates (3) was developed for microgram quantities of brain and required a special adapter for the Beckman spectrophotometer to handle volumes of 40 µ\. or less. The present studies were designed to utilize the greater sensitivity of the ascorbic acid method for the determination of phosphorus in smaller quantities of whole blood, plasma, serum, and urine samples while using only conventional cells for the Beckman spectrophotometer or the Bausch & Lomb Spectronic 20 colorimeter. A thorough investigation has been made of the acidity, ascorbic acid concentration, stability of reagents under different conditions, and effect of time and temperature on the color. A comparison with the Fiske and Subbarow method has been made on a large number of serum and urine samples. APPARATUS, REAGENTS, AND SOLUTIONS
Spectrophotometer. Beckman Model DU spectrophotometer with 1-cm. Corex cells. Used for comparison is a Bausch & Lomb Spectronic 20 colorimeter with 1P40 tube and red filter. Reagent C. Mix 1 volume of 6N sulfuric acid with 2 volumes of distilled water and 1 volume of 2.5% ammonium molybdate, then add 1 volume of 10% ascorbic acid and mix well. Prepare
Upper. Lower.
Ascorbic acid reduction Sulfite-aminonaphtholsulfonio acid reduction
Ammon and Hinsberg (7) were the first to report the use of ascorbic acid for the reduction of phosphomolybdate, but it is difficult to calculate from their data the molar absorbance for comparison with other reports. Lowry and associates (3) modified the previous method by using a stronger ascorbic acid solution with a much longer time of heating at 37° C. and reported a molar absorbance of 25,000 as compared to 4000 for the Fiske
fresh each day. Ascorbic acid, 10%. Dissolve 10 grams of ascorbic acid USP (Mallinckrodt) in distilled water and dilute to 100 ml. Store under refrigeration at 2° to 4° C. The solution is stable for about 7 weeks. Ammonium molybdate, 2.5%. Dissolve 2.5 grams of (NH4)6M07O24.4H20 (Baker’s analyzed) in distilled water, and dilute to 100 ml. Sulfuric acid (Baker’s analyzed). Sulfuric acid, 6N. Dilute 18 ml. of concentrated acid to 108 ml. Perchloric acid (Baker’s analyzed), 72%. Hydrogen peroxide, 30% (Eimer & Amend tested purity). Trichloroacetic acid, 10% (Baker’s analyzed). Before using, check the phosphorus content; it is usually below detectable amounts when in the dilutions used for preparing filtrates, even though the stated analysis may indicate detectable quantities. METHODS
Procedure.
Pipet the phosphorus standards, diluted urine, or trichloroacetic acid fil-
ashed sample of biological material, 1756
VOLUME
2 8,
NO. 11, NOVEMBER
1956
1757
trate (blood, plasma, or serum) containing up to 8 of phosphorus into a 15-ml. graduated centrifuge tube and adjust the volume to 4 ml. with distilled water. The reagent blank consists of 4 ml. of distilled water. Pipet 4 ml. of reagent C into each tube, cap with Parafilm, mix, and place rack wdth all tubes in a 37° C. oven, incubator, or water bath for 1.5 to 2 hours. Remove, allow’ a few minutes to cool to room temperature, and read absorbance in Beckman DU spectrophotometer at 820 against the blank. Preparation of Samples. Urine Phosphorus. Dilute urine with distilled water (if clear) or dilute hydrochloric acid (if turbid). If the approximate range is unknown, run several
dilutions.
Inorganic Phosphorus.
Add 0.5 ml. of w’hole blood, plasma, to 2 ml. of 10% trichloroacetic acid. Mix well, centrifuge, and pipet off the filtrate. For routine analysis use 0.5 ml. of filtrate. or serum
ascorbk:
Figure 3.
acid
concentration
Sensitivity of Method and Stability of Color. The sensitivity using ascorbic acid is about eight times that of the Fiske-Subbarow method, as is seen in Figure 1, which shows standard curves comparing the two procedures. Furthermore, the stability of the color development is very good. After 1.5 hours, the color intensity was increased only slightly. Stability of Reagents. Reagent C is unstable and even if kept at 2° C. in the refrigerator will rapidly lose its ability to form a color w’ith phosphorus. In Figure 2 are showm the relative stabilities of Reagent C and ascorbic acid solutions. The 10% ascorbic acid solution can be kept for many weeks at 2° C. in the refrigerator. The 6¿V sulfuric acid and 2.5% ammonium molybdate can be kept at room temperature. Effect of Ascorbic Acid Concentration. As can be seen in Figure 3, the 1% ascorbic acid concentration used is well above that needed for maximum color development in the phosphorus range desired. Effect of Time and Temperature. The color development at room temperature is incomplete even in 3 hours but is complete in 1 hour at 37° C. (see Figure 4). Ammon and Hinsberg found 7 minutes at 37° sufficient in their w’ork, while Lowry and associates used 2 hours at 37° C. Effect of Acid Concentration. In this method a rather broad but definite range of acid concentration is permissible. As is evident from Figure 5, blank solutions are reduced by ascorbic
%
Effect of ascorbic acid concentration 1.6 7 of phosphorus
on
Lipide Phosphorus. Extract plasma, serum, or whole blood w’ith alcohol-ether (3 to 1 by volume). Add 0.5 ml. of blood to 9.5 ml. of alcohol-ether and place in a hot w’ater bath at 80° C. until boiling occurs. Remove, cool, and readjust to 10 ml. with alcohol-ether. Ash 5 ml. of filtrate or supernatant liquid as below’.
Ashing Procedure. To determine total phosphorus first a sample of biological material. For lipide phosphorus, evaporate and ash the alcohol-ether extract. For total acidsoluble phosphorus of plasma, serum, or whole blood, ash trichloroacetic acid filtrates (see inorganic phosphorus above). Place the sample to be ashed in a 20 X 150 mm. borosilicate glass test tube, add 4 drops of concentrated sulfuric acid (and 2.0 ml. of concentrated nitric acid if whole blood is to be ashed), and heat the test tubes over a sand bath until w’hite fumes of sulfur trioxide appear. Then add 2 drops of 72% perchloric acid to each tube and heat in a Bunsen flame until the liquid becomes clear. After cooling, add distilled water and adjust the volume to 25 ml. in a volumetric flask. Aliquots can then be taken for analysis. Concentrated (30%) hydrogen peroxide can be used instead of perchloric acid for the final ashing, provided it has been analyzed and found free from phosphorus. Many commercially obtainable c.p. peroxide solutions contain appreciable amounts of phosphorus. Ashing with peroxide is slower and all excess peroxide must be removed by boiling before phosphorus is analyzed.
Figure 4.
ash
Effect of time and temperature color development
on
STUDY OF VARIABLES
Selection of Wave Length. The absorption spectra of the colored phosphomolybdate reduction product was the same as that reported (3), and a wave length of 820 mg was also used in this work. Absorption of the blank vs. distilled water is so low that for routine analysis a blank run is unnecessary.
Figure
5.
Effect of sulfuric acid concentration color development at 37° C.
on
CHEMISTRY
ANALYTICAL
1758
acid concentration of 0.4ÍV. Above l.OiV, no reeven in the presence of phosphorus. Within the range 0.5 to LOW, color development is proportional to phosphorus concentration. The value of 0.6W was chosen to eliminate need for neutralizing the acids that may be present from the ashing procedure. The acidity of approximately IN used by Ammon and Hinsberg and by Lowry and associates is at the upper limit. acid below
duction
an
occurs
Table I.
Comparison between Ascorbic Acid and
Aminonaphtholsulfonic Acid Reductions
on
Plasma
(Values indicate mg. of P per 100 ml. of plasma)
Ascorbic
ANSA
Ascorbic
ANSA
Ascorbic
ANSA
2.54 3.03 6.79 3.57 3.01 2.63 2.57 2.66
2.75 3.07 6.78 3.71 2.99 2.71
3.00 3.79 3.36 3.37 3.65 4.82 3.95 3.92 4.06 3.29
2.74 3.67 3.36 3.30 3.55 4.28 4.01 3.98 4.11 3.36
3.32 3.69 2.65 3.92 3.73 3.93 3.62 3.15 3.22 3.62
3.34 3.66 2.68 3.82 3.82 4.07 3.66 3.13 3.34
2.12
1.87
2.61
2.81 2.13 1.90
Av. diff. ± std. diff.
=
—
3.68
acid is used. After dilution, aliquots containing 0.05 ml. or less are contained in the final tube, which is much less than the 0.4W 0.088 ml. of concensafety factor allowed. 3.2 meq. of acid trated sulfuric acid in 8-ml. final volume. The presence of arsenate interferes with phosphorus determination, as it also forms a colored reduction product (1) with molybdate. Eight micrograms of arsenic are equivalent to 1 7 of phosphorus, and the absorbance is proportional to its concentration. Silicate in amounts up to 1 mg. has no effect on phosphorus determination. Although the method as described is usable with 0.5 7 of phosphorus in 4 ml. or 0.1 7 per ml., by slight modification it is adaptable to smaller total amounts. By using 1.5 ml. of trichloroacetic acid filtrate and 1.5 ml. of reagent C, 0.15 7 of total phosphorus can be accurately determined without the use of microcuvettes. A wave length of 820 µ precludes the use of most colorimeters. The Bausch & Lomb Spectronic 20 colorimeter with 1P40 phototube and red filter may be used in this range and was compared with the Beckman DU instrument. An absorption maximum at 820 µ was obtained and standard curves were linear as in the Beckman. However, absorbance was higher for a given solution in the Spectronic. Thus 8 7 of P S 1.0 (Spectronic) vs. 0.850 =
(Beckman).
0.006 ± 0.025.
Besides filling the need for a more sensitive phosphorus micromethod not requiring special microtechniques, this procedure also possesses the advantages of stability, constancy, and linearity.
Quantities of trichloroacetic acid up to four times that normally used for protein precipitation had no effect on the results. In the dilutions used such quantities would not appreciably change a 0.6-Y acid solution, so the results indicate no interference
Table II.
from the trichloroacetate ion.
Normal Values for Whole Blood Phosphorus Determined by Ascorbic Acid Method Mg. of F per 100 Ml, of Blood
COMPARISON WITH
FISKE-SUBBAROW
METHOD
A series of 30 blood plasmas and 20 diluted urines was analyzed for inorganic phosphorus by the Fiske-Subbarow method and by the suggested procedure. The plasma samples were obtained clinically and represent abnormal as well as normal values expressed as milligrams per 100 ml. in Table I. Urine values were obtained strictly for comparing the two methods and the dilutions were varied from 1:100 to 1:200 to bring the concentration of phosphorus within the proper range. The data were treated statistically and the application of the t test indicated that the methods gave identical results. The paired analyses of urine specimens showed an average difference of 0.03 ± 0.04 per ml. TYPICAL
BLOOD VALUES
A number of normal whole blood samples were analyzed by the procedures given for total blood, blood inorganic, blood acidsoluble, and blood lipide phosphorus. Typical values are listed in Table II. DISCUSSION
The final concentration of 0.5 to LOW acid (exclusive of the IN due to ascorbic acid) showed that the color development was proportional to phosphorus concentration. As reagent C, on final dilution, is 0.6W sulfuric acid, the sample itself may contain some acid, which may be trichloroacetic acid in plasma filtrates or sulfuric and perchloric acids from ashing. In all routine analysis, no neutralization of trichloroacetic acid filtrates or acid from ashing is necessary. For example, the addition of 0.5 ml. of trichloroacetic acid filtrate in an 8-ml. final volume would give only 0.03W acid. In ashing, usually less than 0.5 ml. of concentrated sulfuric acid and perchloric 0.
Inorganic
soluble
Lipide
Total
3.71
26.3
10.5 11.3 11.4
31.0 31.0
3.21
2.48 2.96 3.21
3.30 2.84 3.32
23.7 21.9 23.3 23.6 22.2
22.5 25.8
12.7 10.9 10.2
31.6
36.8 31.1
35.0 32.7
33.0 34.6
Stability of the color developed eliminated the necessity of reading solutions immediately or at a certain time after starting a color reaction. Constancy meant that with different batches of ascorbic acid and ammonium molybdate, 8 7 of phosphorus always read 0.840 to 0.850 on several Beckman DU spectrophotometers. Standard curves were always linear over the entire range of the Beckman instrument. For ordinary analysis requiring an accuracy within ±2%, blanks and standards need not be run each time. In the Fiske and Subbarow procedure (2) the absorbance of the blank vs. distilled water as well as the slope of the standard curve may vary from day to day.
LITERATURE
CITED
(1) Ammon, R., Hinsberg, K., Z. physiol. Chem. 239, 207 (1936). (2) Fiske, C. H., Subbarow, Y., J Biol. Chem. 66, 375 (1925), (3) Lowry, O. H., Roberts. N. R., Leiner, K. Y., Wu, M. L., Farr, A. L„ Ibid., 207, 1 (1954). Received for review April 26, 1956. Accepted July 12, 1956. Based on work performed under contract with the U. S. Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, N. Y.
