PROTEIN TURBIDITY PRODUCED BY TRICHLORACETIC ACID AND SULFOSALICYL1C ACID AT VARYING TEMPERATURES AND VARYING RATIOS OF ALBUMIN AND GLOBULIN

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1 THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 44, 1V0. 6 Copyright 1965 by The Williams & Wilkins Co. Primed in U.S.A. PROTEIN TURBIDITY PRODUCED BY TRICHLORACETIC ACID AND SULFOSALICYL1C ACID AT VARYING TEMPERATURES AND VARYING RATIOS OF ALBUMIN AND GLOBULIN HENRY SCHRIEVER, M.D., AND S. RAYMOND CAMBINO, M.D. Department of Pathology, Englewood Hospital, Englewood, A r ew Jersey Henry 1 and Muelemans 2 have recommended the use of trichloracetic acid (TCA) instead of sulfosalicylic acid (SSA) for the turbidimetric determination of cerebrospinal fluid (CSF) protein. TCA is recommended because turbidity is not altered by changes in the albumin/globulin ratio of cerebrospinal fluid. SSA turbidity, on the other hand, is twice as great with albumin as it is with globulin. 1 We have confirmed this particular advantage of TCA over SSA, but find it limited to the narrow temperature range of to 25 C. In addition, this paper details the temperature sensitivity of TCA and SSA turbidity and describes the differential turbidity produced by albumin and globulin with TCA and SSA. MATERIALS 1. Sulfosalicylic acid, 5.0 per cent (w/v). Dissolve 50 Gm. of sulfosalicylic acid (AR crystals) in distilled water and bring to 1000 ml. 2. Trichloracetic acid, 8.0 per cent (w/v). Dissolve 30 Gm. of trichloracetic acid (AR crystals) in distilled water and bring to 1000 ml. 3. Standard albumin solution, 10 mg. of protein nitrogen per ml. Armour standardized albumin, lot Y 3111.* Dilutions were made with distilled water. 4. y-globulin solution. Pooled human serum was fractionated with a mixture containing.8 per cent sodium sulfate and 7.0 per cent sodium sulfite according to Reinhold. 4 The precipitate was dialyzed against Received, March 4, Dr. Schriever's present address is U.S.A.F. Hospital Kecsler, Keesier Air Force Base, Mississippi. Requests for reprints should be sent to Dr. Gambino. * Armour Pharmaceutical Co., Kankakee, Illinois. G67 M/15 phosphate buffer, ph 7.4, at 4 C. for 3 days. Cellulose acetate electrophoresis showed the redissolved precipitate to be 7-globulin. Dilutions were made with 0.9 per cent (w/v) saline solution and were calibrated by the method of Papadopoulos, 3 using Armour albumin as a standard. 5. Lyophilized control serum. Protein concentration 6.9 Gm. per 100 ml. Albumin/ globulin ratio 1.7/1. Versatol, lot t METHODS The TCA turbidity test was performed by mixing 5.0 ml. of 3.0 per cent (w/v) TCA with 1.0 ml. of a standard protein solution and allowing the mixture to stand for 10 min. The SSA turbidity test was performed by mixing 5.0 ml. of 5 per cent (w/v) SSA with 1.0 ml. of a standard protein solution and allowing the mixture to stand for 5 min. All readings were made with a Coleman Jr. Model 6A spectrophotometer,! using 19- by 150-mm. cuvets at 450 rmt. The temperature of all reagents, pipets, and cuvets was controlled with a large bath brought to the desired temperature by mixing cold and hot water in varying proportions. TCA and SSA turbidities were compared at and at 5 C. In addition, albumin, globulin, and control serums were mixed with TCA at various temperatures, and turbidity was read at each of these temperatures: 5, 10, 15,, 23, 25, 30, and 37 C. A fixed protein concentration of 62.5 mg. per 100 ml. was used in this study. Mixtures of pure albumin and globulin were studied with TCA at 5, 23, and 37 C. Four separate A/G ratios were simulated: 2.0/1, 1.0/1, 0.4/1, and 0./1. Predicted turbidities for these artificial mixtures were t Wamer-Chilcott, Morris Plains, New Jersey. t Coleman Instrument Co., Maywood, Illinois.

2 668 SCHBIEVER AND GAMBINO Vol. 44 calculated from data obtained with pure solutions of albumin and globulin at the 3 temperatures. Finally, the protein concentration of 13 CSF specimens was measured, using TCA and SSA at 15, 23, and 30 C. RESULTS 1. TCA turbidity at Three separate concentrations of albumin, 7-globulin, and control serum were mixed with TCA at The resulting turbidities are shown in Figure 1. Albumin and control serum have identical turbidities, whereas 7-globulin has slightly less turbidity. TCA turbidity is not seriously affected by changes in albumin/globulin ratios when studies are made at 2. SSA turbidity at A study similar to that reported under "1" was made with SSA instead of TCA and the results are shown in Figure 2. The turbidity with albumin is nearly twice as great as the turbidity obtained with the control serum, and the turbidity obtained with 7-globulin is less than half that obtained with control serum. SSA turbidity, in contrast to TCA, is seriously affected by changes in albumin/globulin ratios when studies are made at The results of these studies at with TCA and SSA are in agreement with previous reports. 1 ' 2 3. TCA turbidity at 5 C. A study similar to that reported under "1" was made with TCA at 5 C. instead of 23 C, and the results are shown in Figure 3. Again we find that turbidities with albumin and control serum are nearly identical, but this time the turbidity with 7-globulin is greatly reduced. 4. SSA turbidity at 5 C. A study similar to that reported under "3" was made with SSA instead of TCA, and the results are shown in Figure 4. SSA turbidity is still greater with albumin than with control serum, but not nearly as much as at 23 C, and both turbidities are much less than those found at On the other hand, the turbidity produced by globulin at 5 C. is very similar to that produced at Thus, at 5 C. there is little difference between the turbidity produced by the control serum and that produced by 7-globulin. 5. Effect of temperature on TCA turbidity. Table 1 lists the optical densities of albumin, 7-globulin, and control serum standards (each at concentration of 62.5 mg. per 100 ml.) at 8 temperature increments between 5 and 37 C. Above 25 C. the TCA turbidity of albumin is considerably greater than the TCA turbidity of control serum. At 37 C. the TCA turbidity of albumin is almost 3 times as great as the TCA turbidity of control serum. Pure 7-globulin produces less tur- TCA 23 C.4 00 OGAMMfl GLOBULIN AL8UMIN ^CONTROL SERUM uj.j 00 Q y Q. o.0 PROTEIN PER 100 ML FIG. 1. The turbidity of 7-globulin, albumin, and control serum with trichloracetic acid (TCA) at

3 Dec PROTEIN TURBIDITY 6(>9 -SSA 23 C ALBUMIN * CONTROL SERUM o GAMMA GLOBULIN Q < U P.2 00 a O.100 SO ISO MG PROTEIN PER 100 ML FIG. 2. The turbidity of 7-globulin, albumin, and control serum with sulfosalicylic acid (SSA) at >- h- Q TCA 5 C ogamma GLOBULIN ALBUMIN A CONTROL SERUM < O a.0 o 50 MG 75 PROTEIN PER 100 ML FIG. 3. The turbidity of -/-globulin, albumin, and control serum with trichloracetic acid (TCA) at 5 C. 0 bidity than either albumin or control serum, especially at 5 C. 6. TCA. turbidities of prepared albumin/yglobulin mixtures. Table 2 lists the optical densities of prepared albumin/vglobulin mixtures and compares them with predicted turbidities calculated from the turbidity of pure solutions. The predicted values are lower at 5 and 23 C, and are higher at 37 C. The closest agreement occurs at 7. TCA and SSA turbidity of CSF specimens. Table 3 is a summary of estimated protein concentrations obtained for 13 CSF specimens using TCA at 15, 23, and 30 C. In each instance the optical densities were converted to mg. per 100 ml. of protein, using a single calibration curve made with control serum at The estimated concentrations at 15 C. were lower and those at 30 C. higher than concentrations obtained at 23 C, the

4 670 SCHRIEVER AND GAMBINO Vol. U z Ul Q.300 -J < SSA 5* C AL8UMIN ACONTROL SERUM OGAMMA GLOBULltf MG PROTEIN PER 100 ML FIG 4. The turbidity of -/-globulin, albumin, and control serum with sulfosalicylic acid (SSA) at 5 C. TABLE 1 THE EFFECT OF TEMPERATURE ON THE TUKBIDITY OF EQUIVALENT CONCENTRATIONS (62.5 MG. PER 100 ML.) OF ALBUMIN, Y-GLOBULIN, AND CONTROL SERUM, USING TRICHLORACETIC ACID Temperature C Control serum C Optical Density Albumin y-globulin temperature at which the calibration curve was made. Table 4 lists estimated protein concentrations obtained for the same 13 CSF specimens, using SSA instead of TCA at 15, 23, and 30 C. Again a single calibration curve was used, made with control serum at With SSA the estimated concentrations at 15 and 30 C. are not very different from those obtained at Thus, CSF turbidity produced with SSA is less temperature-dependent than CSF turbidity produced with TCA. Polyacrylamide gel "disk" electrophoresis of each of the 13 CSF specimens showed them to be mixtures of albumin and globulin, with albumin predominating in all. This accounts for the tendency of the SSA estimates of protein to be slightly higher than those obtained with TCA at 23 C, as SSA yields more turbidity with albumin. DISCUSSION Because TCA produces about the same turbidity with albumin as it does with globulin, whereas SSA does not, it would seem that TCA is preferable to SSA for the measurement of protein by turbidity. Unfortunately, this advantage of TCA over SSA is limited to temperatures between and 25 C. Above 25 C. TCA begins to react like SSA, producing much more turbidity with albumin than with globulin. If TCA is used in laboratories with uncontrolled room temperature, the accuracy of protein estimates will be decreased. On hot days falsely high values will occur, and if ice-cold TCA reagent is used, falsely low values will be seen. A similar situation holds for SSA, but the temperature effect is less marked. The effect of temperature on turbidity and the differential turbidity produced by albumin and globulin must be controlled if accurate protein estimates are to be made with

5 Dec PROTEIN TURBIDITY 671 TABLE 2 TRICHLOKACETIC ACID TURBIDITIES OF PREPARED MIXTURES OF ALBUMIN AND -^-GLOBULIN COMPARED WITH TURBIDITIES PREDICTED BY CALCULATION Protein Concentration A/G* Found O.D.t at 5 C. Predicted Found 0. D. at Predicted Found O.D. at 37 C. Predicted mg./wo ml /1 1.0/1 0.45/1 0./ G S S S * Albumin/globulin ratio. f Optical density. TABLE 3 KESULTS OF 13 CEREBROSPINAL FLUID (CSF) PROTEIN DETERMINATIONS AT 3 DIFFERENT TEMPERATURES USING TRICHLORACETIC ACID* TABLE 4 RESULTS OF 13 CEREBROSPINAL FLUID (CSF) PROTEIN DETERMINATIONS AT 3 DIFFERENT TEMPERATURES USING SULFOSALICVLIC ACID* Specimen No. 15 C. CSF Protein 30 C. Specimen No. 15 C. CSF Protein 30 C mg./looml I S 52 mg./loo ml. 39 2S S * Control serum calibration curve prepared at SSA or TCA. The use of human or bovine albumin as a standard for turbidimetric protein determinations should be abandoned. If albumin is used as a primary standard with SSA, false estimates of protein concentration in albumin/globulin mixtures will be obtained. If albumin is used as a primary standard with TCA, false estimates will occur unless temperature is controlled. The proper standard for calibrating TCA and SSA turbidity tests is a standard whose albumin and globulin concentration is near that of the material to be measured. Use of * Control serum calibration curve prepared at human serum or CSF standards would lead to greater accuracy in estimating CSF protein concentrations with either TCA or SSA. Finally, greater attention must be paid to temperature. Calibration curves and sample measurements must be made at exactly the same temperature. SUMMARY Trichloracetic acid (TCA) is theoretically superior to sulfosalicylic acid (SSA) for turbidimetric estimates of protein concen-

6 672 SCHKIEVER AND GAMBINO Vol. U tration because TCA turbidity is less affected than SSA turbidity by variations in albumin/globulin ratios. Unfortunately, this advantage of TCA is limited to a temperature range between and 25 C. TCA turbidity, even more than SSA turbidity, is greatly affected by changes in temperature. Therefore, calibration curves and sample measurements must be made at the same temperature. Albumin is not recommended as a primary standard for turbidity tests using TCA or SSA. Human serum or cerebrospinal fluid standards are superior. REFERENCES 1. Henry, R. J., Sobel, C, and Segalove, M.: Turbidimetric determination of proteins with sulfosalicylic and trichloroacetic acids. Proc. Soc. Exper. Biol. & Med., ft?: 748-7, Meulemans, 0.: Determination of total protein in spinal fluid with sulfosalicylic acid and trichloroacetic acid. Clin. Chim. Acta, 5: , Papadopoulos, N. M., Hess, W. C, O'Doherty, D., and McLane, J. E.: A procedure for the determination of cerebrospinal fluid total protein and gamma globulin in neurologic disorders. Clin. Chem., 5: , Rheinhold, J. G.: In Reiner, M.: Standard Methods of Clinical Chemistry, Vol. 1. New York: Academic Press, Inc., 1953, pp