Sep 11, 1973 - A New Micromethod for Determination of Protein in ... This paper presents a new method for determina- .... method, and by the Kjeldahl method.
CLIN.
CHEM.
19/11,
1265-1267
(1973)
A New Micromethod for Determination of Protein in Cerebrospinal Fluid and Urine Michael A. Pesce and Carl S. Strande We developed a new micro-scale procedure for determination of protein in cerebrospinal fluid and urine. The sample (50 l) is mixed with a trichloroacetic acid-Ponceau S dye solution. The proteins, together with a proportional amount of dye, are precipitated and the red precipitate is dissolved in dilute sodium hydroxide solution, giving a violet-colored solution. This color, linearly proportional to the amount of protein present (up to 150 mg/dl), is measured spectrophotometrically at 560 nm. By varying the sample size alone, protein concentrations up to 1500 mg/dl can be measured. The detection limit for the method is 2 mg/dl of sample. Precipitation of the proteins is independent of temperature and albuminbinding compounds, and is not appreciably affected by the albumin-gamma globulin ratio. Additional Keyphrases: dye-protein complex, spectrophotometry of #{149} inter-method comparison One of the most common means for the estimation of protein in cerebrospinal fluid (CSF) is turbidimetry. In both trichloroacetic acid (TCA) and sulfosalicylic acid (SSA) turbidimetric methods, temperature plays a crucial role (1). As the temperature is increased, turbidity increases. In the SSA method, the turbidity is significantly affected by the ratio of albumin to gamma globulin, albumin yielding more turbidity than globulin per unit of weight (2). Up to now, we have used the Lowry method (3) for the determination of protein in CSF. This reaction, although mqre sensitive than the biuret method, is not specific because CSF contains many colorproducing nonprotein substances (4). The biuret method (5), widely used for the determination of protein in urine, depends on the formation of a colored chelated complex between cupric ions and peptide bonds. This paper presents a new method for determination of protein in CSF and urine, based upon coprecipitation of protein and Ponceau S dye by TCA, dissolution of the precipitate in dilute alkali, and spectrophotometric determination of the dye in alkaline solution.
Materials
and Methods
Reagents 1. Ponceau S stock solution. ceau S dye (Aldrich Chemical
Dissolve 40 g of PonCo., Cedar Knoll, N.
From the Columbia Presbyterian Medical Center, Babies’ pital, Clinical Chemistry Laboratory, 3975 Broadway, New N. Y. 10032. Received July 5, 1973; accepted Sept. 11, 1973.
HosYork,
J.) in 1 liter of distilled water. 2. TCA-Ponceau S concentrated reagent. Dilute 20 ml of reagent 1 to 1 liter with TCA solution (300
g/liter). 3. TCA-Ponceau S normal working reagent. Dilute 100 ml of reagent 2 to 1 liter with distilled water. This solution is stable for several months at room temperature. 4. “Albustix” strips (Ames Co. Div. of Miles Laboratories, Elkhart, md.). 5. Sodium hydroxide, 8.0 g/liter. 6. Protein stock standard, human protein standard, 8.0 g/dl (Dade Reagents, Inc., Miami, Fla). 7. Protein working standards, dilute reagent 6 to 40 and 80 mg/dl with distilled water. Procedure (a) Apply sample (CSF or urine) to the Albustix strip, to determine the approximate protein content. If the Albustix reading is: (1) less than 100, use 50 til of sample; (2) between 100 and 300, use 25 Ml of sample and multiply the results by two; (3) between 300 and 1000, use 5 il of sample and multiply the results by 10. (b) To 50 l of each standard and the proper amount of sample, add 500 pl of reagent 3. (c) Mix well; centrifuge for 10 mm at 3500 rpm and completely remove the supernatant liquid with a Pasteur pipet. To the red precipitate add 1 ml of reagent 5, mix and read at 560 nm against water. The color is stable for 6 h at room temperature. Calculate the results by using the Lambert-Beer equation. (d) For samples having protein concentrations of less than 10 mg/dl, the procedure is modified by using 500 id of sample, 50 l of reagent 2, and continuing from part c.
Results and Discussion A bsorption spectrum. We measured the absorption spectrum for a CSF protein-Ponceau S dye complex, a urine protein-Ponceau S dye complex, and an albumin standard-Ponceau S dye complex. The measurements were made against a water blank in a 10-mm cuvette. In all cases, the maximum absorption is at 560 nm. Linearity. The standard curve (Figure 1) is linear from 10 to 150 mg of protein per deciliter. A linear relationship exists for the modified procedure for samples ranging from 2 to 10 mg of protein per deciliter. To establish optimum Ponceau S dye conditions, we examined several concentrations of PonCLINICAL
CHEMISTRY,
Vol.
19, No.
11, 1973
1265
.60
Table 1. Some Representative Values for Estimates of Albumin-Globulin Ratios in 40 mg/dl Albumin-Gamma Globulin Mixtures
.52
Absorbance .44
Albumin in sample, %
100 90 80 70 60 50 40 30 20 10 0
.36 A .28
.20
.12
.04 20
60
ALBUMIN
00
curve
0.154 0.153 0.146 0.145 0.147 0.147 0.146 0.137 0.137 0.139 0.138
SSA.PonceauS dye reagent
Lowry method
0.167 0.165 0.157 0.142 0.139 0.134 0.129 0.128 0.129 0.123 0.116
0.105 0.104 0.109 0.115 0.121 0.121 0.124 0.128 0.133 0.142 0.148
60
Table 2. Results of Protein Determinations Three Different Temperatures
STANDARDS mg/dl
Fig. 1. Standard mg/dl
TCA-PonceauS dye reagent
for concentrations
of
Experimentally determined mg/dl, at temperatures
10-150 Albumin standard, mg/dI
Poncea 8mg/di
20 40 80 100 160
at
values, of
0#{176}C
24#{176}C
37#{176}C
21 42 79 98 157
21 40 79 101 158
22 41 81 97 158
Poncea
4mg/di
ries of standards, CSF, and urine. The mean coefficient of variation was 2.5% (Table 4). Comparison with other methods. Results of the 2mg/di present method plotted against those obtained by the TCA-turbidimetric method yielded a correlation coefficient of 0.726 and a linear regression equation: y = -0.558 + 0.925x. Statistical analysis of the paired data (TCA-turbidimetric method minus present method) gave a mean deviation of 5.143 and a standard deviation of 4.178, yielding a r value of ALBUMIN STANDARDS 4.688 (n = 70), which shows that the bias between leg/dI methods is statistically significant. Fig. 2. The effect of dye concentrations on linearity Results by the present method vs. those by the ceau S dye in TCA solution (30 g/liter). As shown in Lowry method had a correlation coefficient of 0.967 Figure 2, linearity was achieved at a Ponceau S dye and the linear regression equation: y = -12.276 + concentration of 8 mg/dl. 0.998x. Statistical analysis of the paired data Albumin-gamma globulin effect. As shown in (Lowry method minus present method) gave a mean Table 1, the color produced by varying the albumin deviation of 12.412 and a standard deviation of and gamma globulin concentration is least affected 11.489, yielding a r value of 6.299 (n = 34), which shows that the bias between the methods is again by the TCA-Ponceau S dye reagent. Temperature. Results obtained when the TCAstatistically significant. Ponceau S dye reagent is added to a series of albuJudged from the paired difference analysis, the min standards at temperatures of 0, 24, and 37 #{176}C present method gives values that are lower and stawere identical (Table 2). tistically different from both the TCA-turbidmetric Recovery and precision. Recovery data for albumin method and the Lowry method. The Lowry method and gamma globulin added to CSF and urine are is subject to interference from drugs and free amino shown in Table 3. Within-run precision was meaacids (4), and thus gives erroneous results. To acsured by performing replicate determinations on alcount for the differences obtained between our methbumin standards, 15 pooled CSF samples, and on od and the TCA-turbidimetric method, we analyzed three pooled samples of urine. The mean coefficient CSF samples for protein by our method, the TCAof variation was 2.0% (Table 4). Day-to-day precision turbidmetric method, and by the Kjeldahl method. was measured over a 12-day period on the same seThe results (Table 5) indicate good correlation beA
1266
CLINICAL
CHEMISTRY,
Vol. 19, No. 11, 1973
Table 3. Recovery of Albumin Protein in CSF
and Gamma Globulin Added to CSF and Urine Gamma globulin added
Albumin added
Protein in urine
23 23 23 23 75 75 75 75
20 40 10 20 19 100 20 40 32 32 32 32 87 87 87 87
10 80 10 40 20 100 20 80
Found
Expected
43 62 32 40 91 178 89 105 43 107 42 66 107 177 102 155
43 63 33 43 94 175 95 115 42 112 42 72 107 187 107 167
Recovery,
V
100 98 90 85 84 103 70 75 110 94 100 85 100 90 75 85
(amt. found of added/amt. added) X100. Values are in mg/dl.
Table 4. Precision for Protein Determinations in CSF and Urine Mean Sample
n
SD
mg/dl
CV,
%
Table 6. Results of Protein Determinations in an Albumin Standard Containing Abnormally High Concentrations of Su Ifisoxazole, Sal icyl ic Acid, Sulfadiazine, and Bilirubin Concentration
Within-run
40 mg/dl std. 80 mg/dl std. Pooled CSF Pooled urine
25 25 25 25
40 79 53 57
1.7 1.5 2.5 2.3 2.0
0.68 1.15 1.34 1.32 Mean
Substance
Su lfisoxazole Salicylic acid
Day-to-day 40 mg/dl std. 80 mg/dl std. CSF pool Urine pool
12 12 12 12
41 78 55 54
0.82 1.35 1.58 1.91 Mean
2.0 1.7 2.9 3.5 2.5
Table 5. Protein Content of CSF Samples, as Determined by TCA-Tu rbid imetric Method, Present Method, and Kjeldahl Method TCA-Turbldimetric
68 68 92 73
Present mg/dI
of
substance, mg/dt, in 80 mg/dl albumin standard
Sulfadiazine Biliru bin
Concentration of solution, mg/dl
0
80
50
78
100 50 100
78 80 81
50 100
79 81
2 4
81 79
mg/dl can be detected, this method be measured in normal urine.
allows protein
to
Kjeldahl
48 55 71 51
46 56 71 53
tween our method and the Kjeldahl method. Several compounds that bind to albumin were tested for possible interference with our method. As shown in Table 6, these substances cause no alteration in the binding of the Ponceau S to the protein. There is no upper limit to the protein concentration that can be measured. By using a S-gil sample, protein concentrations of 1500 mg/dl can be measured; samples with protein concentrations of greater than 1500 mg/dl can be diluted and then measured. Since protein concentrations as low as 2
We thank terminations.
Dr. Calvin
L. Long
for performing
the
Kjeldahl
de-
References I. Schriever, H.. and Gambino, S. R., Protein turbidity produced by trichloroacetic acid and sulfosalicylic acid at varying temperatures and varying ratios of albumin and globulin. ,4mpr. .1. (‘fin. Pat hot. 44, 667 (1965). 2. Meulemans, 0., with sulphosalicylic Acta 5,757(1961).
Determination of total protein in spinal fluid acid and trichloroacetic acid. C/in. Chim.
3. Lowry. 0. H., Rosebrough, N. ‘1.. Farr, A. L., and Randall, R. J., Protein measurement with the Folin-phenol reagent. J. Rio!. Chem. 193,265(1951). 4. Zondag, H. A., and Van Boetzelaer. G. L., Determination of protein in cerebrospinal fluid-sources of error in the Lowry method. (‘un. (him. Acta 5, 155(1960). 5. Kingsley, C. R., The direct hiuret method for the determination of serum proteins as applied to photoelectric and visual colorimetry. J. Lab. (‘tin .Md. 27, 840 (1942). CLINICAL
CHEMISTRY.
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