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MECHANISM AND SIGNIFICANCE OF THE THYMOL TURBIDITY TEST FOR LIVER DISEASE Henry G. Kunkel, Charles L. Hoagland J Clin Invest. 1947;26(6): Research Article Find
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MECHANISM AND SIGNIFICANCE OF THE THYMOL TURBIDITY TEST FOR LIVER DISEASE Henry G. Kunkel, Charles L. Hoagland J Clin Invest. 1947;26(6): Research Article Find the latest version: MECHANISM AND SIGNIFICANCE OF THE THYMOL TURBIDITY TEST FOR LIVER DISEASE By HENRY G. KUNKEL AND CHARLES L. HOAGLAND' (From the Hospital of the Rockefeller Institute for Medical Research, New York City) (Received for publication April 7, 1947) An increase in the amounts of the globulin components of the serum has long been recognized in advanced states of liver disease (1, 2, 3), although the significance of the alteration has never been defined. It has been the basis of the Takata-Ara, Weltmann and the formol-gel reactions which have been used for many years in the diagnosis of liver disease. The nonspecific nature of these tests is now clear following the demonstration that positive reactions are found in any disease showing marked hyperglobulinemia (4, 5, 6). More recently, several new serum reactions, which appear to depend on small changes in the proteins of the serum, have been used for demonstrating liver disease. These include the cephalin flocculation, the colloidal gold and the thymol turbidity tests. The cephalin flocculation reaction has been studied in detail by Hanger and his coworkers (7, 8) and has been found to be a sensitive index of liver damage. The test may be positive in patients with liver disease who show normal serum protein values according to the usual methods of protein estimation and, conversely, the serum of patients with marked hyperglobulinemia may show negative cephalin flocculation reactions. The exact serum protein constituent that is altered in liver disease and is responsible for a positive cephalin flocculation test has not been clearly established; recent work by Hanger (8) appears to implicate albumin in addition to gamma globulin. The final solution to the problem is hampered by the complexity of the cephalin flocculation reaction, a fact which is also true of the colloidal gold reaction. The technique of the thymol turbidity test is much simpler, however, and probably consists of a direct precipitation of a protein appearing in liver disease by the addition of a thymol solution. It would seem, therefore, that a study of the mechanism of this reaction and the protein com- Deceased, August 2, ponent concerned would be more likely to yield clear-cut information regarding at least one of the proteins that appear in the blood stream during diseases of the liver. The present study was an attempt to elucidate the mechanism of the reaction and to define its significance in terms of clinical observations. MATERIALS AND METHODS The sera used in the study of the thymol turbidity reaction were selected from a group of 200 patients with infectious hepatitis and 65 patients with other liver disorders admitted to the Out Patient Department of the Hospital of the Rockefeller Institute. Technique- of performing the thymol turbidity test. The thymol reagent was prepared as described by Maclagan (9). Slight variations of this method produced unsatisfactory results. In alkaline solution thymol is somewhat unstable and turbidity of the reagent often occurs on standing. Exposure to air increases the turbidity of the solution and it was found important to keep the thymol reagent tightly stoppered. As the solution becomes increasingly cloudy, its activity decreases and it is important that only clear or very slightly turbid solutions be used. If properly prepared, the thymol reagent is satisfactory for at least one month. Although the ph of the thymol reagent was slightly lower than that originally stated by Maclagan, it proved to be satisfactory. Three ml. of the thymol reagent were added to 0.05 ml. of serum and the degree of turbidity measured in the Coleman Jr. spectrophotometer at 650 mnu. This represented a 1/60 dilution and corresponded to that originally described by Maclagan. Figure 1 shows the turbidity of sera from cases of liver disease at various dilutions of reagent using saline dilutions as controls. It can be seen that the maximum turbidity was usually obtained at a 1/12 dilution and that differences in certain sera at the lower dilution might not be so marked at the 1/60 dilution. Although the use of lower dilutions has certain apparent advantages, all determinations discussed in this paper were performed at Maclagan's standard 1/60 dilution. The degree of turbidity was compared with a BaSO4 standard as described in a previous publication (10). This proved to be a satisfactory standard regardless of tube size or type of instrument used. The units of turbidity corresponded to those originally' described by Maclagan using visual comparators. 1060 THYMOL TURBIDITY TEST IN LIVER DISEASE 1061 ' 30 I l dll J 50 a10, in 60 eal e 60o 2 o o PN BFamt thymol Peagent added to one part zerum FIG. 1. TURBIDITY READINGS AS MEASURED IN THE SPECTROPHOTOMETER AT VARIOUS DILUTIONS OF SERUM WITH THYMOL REAGENT Estimation of the degree of flocculation of the thymol serum mixture was also carried out in a number of patients. The finding of Neefe (11) that in certain patients, following an attack of infectious hepatitis, the 24-hour flocculation with thymol reagent remained posi-. tive for a slightly longer period than the usual thymol turbidity reaction, was confirmed. In general, however, the estimation of flocculation proved to be a less sensitive index than turbidity determinations, and did not furnish quantitative results. Electrophoretic analyses were made in diethylbarbituric acid buffer (.u = 0.1, ph = 8.6) by the method of Longsworth (12). Determinations of lipids were carried out by the gasometric lipid carbon method of Van Slyke and Folch (13). Total and free cholesterol of the plasma was determined by the method of Schoenheimer and Sperry (14). Extraction of lipids from serum was done by freezing in the presence of ether as described by Mc- Farlane (15). A modification of Hanger's method (7) was used for the determination of the cephalin flocculation reaction. Bromsulfalein retention was estimated by the method of Rosenthal and White (16) modified for the use of the Coleman Jr. spectrophotometer. Globulin was determined electrophoretically and by fractionation with NaSO, (17). In addition, quantitative measurements of globulin were obtained by a turbidometric technique (18). Immunological experiments were carried out by injecting rabbits with 5 to 8 mgm. of thymol protein every 2 days for 8 injections. The antiserum was absorbed with normal human serum. Precipitin tests were carried out by the technique of Swift, Wilson, and Lancefield (19). EXPERIMENTAL I. The relation of the thymol turbidity reaction to the lipids in the serum It was demonstrated by Maclagan (9) that the precipitate resulting from the addition of thymol reagent to serum is high in cholesterol and phospholipids. Recant, Chargaff and Hanger (20) found that sera from cases of liver disease from which lipids had been extracted with ether no longer showed turbidity following the addition of thymol reagent. These observations indicated that lipids are an important factor in the thymol turbidity reaction. This work was confirmed and extended. Thymol was found to have a special effect on lipids in general. Any lipid emulsion tended to be broken up by the addition of a thymol solution. Figure 2 shows the effect of thymol in increasing the particle size of a lipid emulsion as viewed under the microscope. When such emulsions were visualized with the naked eye, an increase in turbidity accompanied the change in particle size. This turbidity was purely the result of the physical alteration in the lipid emulsion resulting from the addition of thymol. Lipemic sera from patients with liver disease, nephrosis, diabetes, and thyroid disease all showed an increase in turbidity upon the addition of the thymol reagent. This, however, was purely a physical change in the lipid emulsion, since no protein was precipitated as in the usual thymol turbidity reaction accompanying liver disease. The following example serves to illustrate this point. Three-tenths ml. of lipemic serum from a patient with nephrosis was diluted 60 times with the thymol barbital buffer reagent. This gave an increased turbidity over 0 0 o o 00 0 al FIG. 2. COMPARISON OF THE PARTICLE SIZE OF EQUAL QUANTITIES OF A LIPID SUSPENSION IN (a) BARBITAL BUFFER, (b) BARBITAL BUFFER PLUS THYMOL, AS VIEWED UNDER THE MICROSCOPE 0 0 b 0( 0 00- 1062 HENRY G. KUNKEL AND CHARLES L. HOAGLAND the serum with barbital buffer alone equivalent to 60 units. The turbid mixture was then spun for 3 hours in the centrifuge at 15,000 r.p.m. No sediment settled at the bottom of the tube, but a white layer formed at the surface leaving a clear solution underneath. Analysis of the surface layer showed that it contained 6 mgm. total lipid and mgm. nitrogen. The material responsible for the turbidity had been brought to the surface of the solution by high speed centrifugation and was found to contain a negligible amount of protein. A similar experiment carried out on the turbid material resulting from the addition of thymol reagent to clear serum from a case of infectious hepatitis demonstrated that the cloudy material all settled to the bottom of the tube. Nitrogen analysis of the precipitate showed that it contained 0.44 mgm. of N. Whereas the turbidity of this serum had been found to be equivalent to only 30 units, the material responsible for the turbidity consisted of a large amount of protein. It appears, therefore, that the turbidity produced by the action of the thymol reagent on lipemic sera from subjects without liver disease is due to an increase in the particle size of the protein-free lipid suspension, while the turbidity produced by the reagent in clear hepatitis serum is due to the formation of a protein-lipid-thymol complex. Since certain sera from cases of liver disease are lipemic, it was of some importance to find a simple method of determining how much of the turbidity produced by the thymol reagent indicated a true reaction with precipitation of protein. When a thymol solution having the same ph as the usual reagent, but with a high ionic strength, was added to clear serum from a patient with liver disease giving a positive reaction, no turbidity appeared. However, when this solution was added to an artificial lipid emulsion or lipemic sera from patients with nephrosis, the turbidity produced was the same as that caused by the low ionic strength reagent. The thymol altered the state of the lipids regardless of the ionic strength of the solution. The usual protein precipitation reaction occurred only with the low ionic strength thymol reagent. In evaluating the turbidity produced in lipemic serum by Maclagan's thymol reagent, the amount of turbidity due to protein precipitation alone was obtained by using as blank in the photometer serum with the high ionic strength thymol buffer (Table I). The presence of lipids in the serum is also an essential factor in the usual thymol turbidity reaction, as indicated by the fact that positively reacting sera after extraction with ether no longer give the reaction. The essential role of the lipids was further borne out by the finding that surfaceacting agents, such as various tweens, completely inhibited the formation of any precipitate in hepatitis serum to which thymol had been added. Once formed, the thymol precipitate also dissolved readily on the addition of small amounts of tween 80.2 In other protein precipitation reactions which depended purely on the natural solubility of the proteins involved, the presence of tween actually enhanced the precipitation. The action of the tween was undoubtedly related to its effect on the state of the lipids involved in the reaction. Since the soluble lipids play an essential part in the thymol turbidity reaction, it seemed important to test the effect of the addition of various concentrations of lipid on the reaction. Because of the specific effect of thymol on lipid suspensions it was important to keep the lipids in their most soluble state. As a result, lipid was added in the form of clear serum giving a negative thymol turbidity reaction. Four such sera were chosen containing varying amounts of lipid. When each of these sera was added to normal serum, the thymol turbidity reaction of the combination remained negative. However, when added to serum with a high gamma globulin level TABLE I Comparison of the turbidity in units obtained in the spectrophotometer for various sera upon the addition of the thymol reagent, using as the zero control the same sera with (a) buffer alone, (b) high ionic strength buffer with thymol Bfeat Buffer + Type of serum ph 7.6 and th7y6 cad Clear serum from patients with infectious hepatitis Lipemic serum from patient with 52 0 nephrosis Lipemic serum from patient with infectious hepatitis giving a positive thymol reaction, the turbidity of the combination was proportional to the amount of lipid in the added serum. This effect was more strikingly brought out by first extracting the 2 Polyoxyethylene- sorbitan monooleate. lipids from the serum of another patient with a high gamma globulin level in the serum. The thymol turbidity reaction was reduced from 22 to 9 units by this procedure. Table II shows the TABLE II Relative effect of ihe addition of 0.2 ml. of sera, differing only in their lipid content, in restoring a positive thymol turbidity reaction to 0.2 ml. ether-extracted hepatitis serum Thymol Lipid Thymol turbidity of content turbidity combination Protein in Lipid in of serum of serum of extracted precipitate precipitate added added hepatitis serum and added serum mgm. units units mgm. mgm. per cent THYMOL TURBIDITY TEST IN LIVER DISEASE relative effectiveness of various sera in restoring a positive thymol turbidity reaction to this extracted hepatitis serum. It can be seen that the high lipid sera had a much greater effect than normal serum. The major portion of the in- 400 C4 U V~~~~~~~R 7A Concentration of lipid in -seum added (m9. 70) FIG. 3. EFFECT OF THE ADDITION OF SERA CONTAIN- ING VARIABLE AMOUNTS OF LIPID ON THE TURBIDITY, LIPID CONTENT, AND PROTEIN CONTENT OF THE PRE- CIPITATE FORMED IN EXTRACTED HEPATITIS SERUM WITH THYMOL REAGENT 1063 creased turbidity was due to increased precipitation of lipid as shown by the protein and lipid analyses of the precipitates. Figure 3 demonstrates more clearly the comparative effect of various lipid concentrations on the turbidity, protein content, and lipid content of the precipitate formed with extracted hepatitis serum. The results were obtained in the same experiment illustrated in Table II. It is evident that, although there is increased precipitation of protein in the presence of higher lipid concentrations, the major portion of the increased turbidity is due to increased precipitation of lipid. In other words, the resulting turbidity reflected primarily the concentration of lipid in the added serum. The relationship was so close that it was possible to use this system as a rapid method of estimating the concentration of lipid in an unknown serum. The above data demonstrated the marked influence of lipid concentration on the thymol turbidity reaction in a somewhat artificial system involving the addition of sera with variable lipid concentrations. In order to evaluate more clearly the effect of the concentration of lipids in sera on the reaction as it is usually applied, lipid analyses were carried out simultaneously on the serum of patients with liver disease and the specific precipitate resulting from the addition of thymol reagent. The amount of lipid in the precipitate varied from 20 to 50 per cent and was directly proportional to the concentration of lipid in the original serum (Figure 4). The turbidity that is usually measured in the thymol turbidity reaction depends on both the protein and lipid that are precipitated. Since the concentration of lipid in the precipitate is proportional to the concentration in the serum, it is clear that the thymol turbidity reaction actually determines in part the concentration of lipid in the serum of patients with liver disease. Despite the fact that the level of the lipids in the serum is one of the variables that is measured in the thymol turbidity reaction, a number of patients with liver disease other than infectious hepatitis have been observed with high lipid levels in the serum but with low or negative thymol turbidity test values. Fractionation of the total lipid in these cases into the cholesterol and phospholipid partitions did not reveal any specific effect. The addition of reactive protein, or serum contain- 1064 IL HENRY G. KUNKEL AND CHARLES L. HOAGLAND 1500 Concentration of lipid in Serum (m9.76) 2000 FIG. 4. THE RELATION OF THE LIPID CONTENT OF SERUM FROM PATIENTS WITH LIVER DISEASE TO THE LIPID CONTENT OF THE SPECIFIC PRECIPITATE FORMED IN THE THYMOL TURBIDITY REACTION ing reactive protein, always produced a positive reaction in proportion to the total lipid concentration. The presence of the thymol-reacting protein was the essential factor while the level of the lipids affected only the intensity of the reaction. II. Identification of the protein involved in the thymol turbidity reaction Maclagan (9) analyzed the precipitate obtained in a positive thymol turbidity reaction and found that it consisted of approximately 40 per cent protein. He suggested that this was probably a gamma globulin because of its low solubility. Hanger and associates (20) were unable to obtain positive thymol turbidity reactions by adding gamma globulin electrophoretically separated from hepatitis sera to normal sera and various lipid fractions. They concluded that the protein involved in the reaction wars probably an alpha or beta globulin and not in the gamma globulin fraction. (a) Electrophoretic analysis of the thymol precipitate. Studies of -the precipitate obtained with thymol reagent were hampered by the large amount of lipid that was present. Turbid solutions were always obtained when attempts were made to dissolve the precipitate and it was impossible to obtain an electrophoretic pattern. Extraction of the lipids from the precipitate in the cold with ether alone, ether and alcohol, or acetone alone, always resulted in denaturation of the proteins in the precipitate. When removed from serum the proteins were apparently less resistant to the action of organic solvents. Attempts were also made to dissolve the precipitate resulting from the thymol reaction in normal serum and observe the change in the electrophoretic pattern. The high lipid concentration, however, still interfered and no conclusive results could be obtained. The difficulty was finally overcome by the use of tween 80, a strong emulsifying agent. A 1 per cent concentration of this material did not affect the electrophoretic pattern of normal serum. A clear solution suitable for electrophoretic determinations was obtained in the following manner. Two hundred ten ml. of thymol reagent were added to 15 ml. of very reactive hepatitis serum (38 units) at 00 C. The precipitate was collected by centrifugation in the cold and suspended in 10 ml. barbital buffer containing 1 per cent tween at ph 8.5 and A = 0.1. This was then dialyzed in the cold against barbital buffer at the same ph and ionic strength. A small amount of sediment that still remained was thrown down by centrifugation and the resulting supernate was quite clear. Figure 5 shows the electrophoretic pattern of the protein solution obtained in the above manner. The sharp peak (b), representing almost the entire amount of protein present, had a mobility of 1.6 x 10-5 which places it in the gamma globulin fraction but with an unusually rapid mobility. A (a) (b) (b) (a) FIG. 5. ASCENDING AND DESCENDING ELECTROPHORETIC PATTERNS OF THE PROTEIN COMPONENT OF THE PRE- CIPITATE FORMED IN THE THYMO
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