INSTANTANEOUS RATE KINETIC METHOD FOR DETERMINING TOTAL PROTEIN IN CEREBROSPINAL FLUID

Abstract: An instantaneous rate kinetic method for determining total protein in cerebrospinal fluid which avoids the generally high results of prior art biuret methods. The method comprises the steps of: (a) reacting the total protein present in said cerebrospinal fluid sample with a biuret reagent to form colored reaction products; and (b) spectrophotometrically measuring the instantaneous rate of change of the formation of the colored products. The spectrophotometric measurement is directly proportional to said total protein concentration in the sample being assayed.

Description: INSTANTANEOUS RATE KINETIC METHOD FOR DETERMINING TOTAL PROTEIN IN CEREBROSPINAL FLUID Background of the Invention 1. Field of the Invention The instant invention pertains to a kinetic method for determining total protein in cerebrospinal fluid and, in particular, to an instantaneous rate kinetic method for determining total protein in cere- brospinal fluid. 2. Description of the Prior Art Henry et al. (1) report three major problems in determining the protein content of cerebrospinal fluid (CSF): (a) the concentration of the protein is low (normally 15-45 mg protein per 100 ml); (b) the concentration of nonprotein interferring substances is high relative to the protein concentration; and (c) the concentration of inorganic ions is high. Henry et al. (1) state that*-most of the methods for determination of protein in serum and plasma have been applied to CSF but have not generally proved satisfactory. In parti¬ cular, the Kjeldahl digestion is frequently used as the reference procedure but there are problems in obtaining an adequate volume of CSF (10-15 ml) and in correcting for the disproportionately large amount of npnprotein present (2, 3). The biuret reaction has also been used (4, 5, 6), but the results obtained are generally high in comparison with other procedures (3, 7, 8). Ac¬ cording to Henry et al. (1), this discrepancy is be- lieved to result from interference by proteoses, pep¬ tones, and polypeptides present in relatively high con- ' centration. Henry et al. (1) state that when a single biuret reagent is used, the spinal solution must be centrifuged before measuring the color to remove the white precipitate of Ca(OH)„ and Mg(OH) 2 that forms. Precipitation can be prevented by the use of the di- sodium salt of ethylenediaminetetraacetic acid (9). Reading of the biuret color at 330 n to increase sen¬ sitivity has also been suggested (5). Only one pro- cedure using the biuret reaction has been reported to give results comparable with those obtained with micro- Kjeldahl and turbidimetric methods (8). In this pro¬ cedure, the CSF protein is precipitated with trichloro- acetic acid, the biuret reaction is performed on the protein precipitate, ^ and the resultant color is read at 330 nm. Direct spectrophotometric ' measurements of protein at 220 or 280 nm (7, 10, 11, 12) are subject to interference by ascorbic acid, tryptophan, nucleosides, peptides, and proteoses resulting frequently in falsely elevated results (7). Before using direct spectropho- etric measurements, it has been recommended that CSF proteins be purified by gel filtration (7). The two most satisfactory techniques for the determination of CSF protein are reported by Henry et al. (1) to be the turbidimetric and Folin-Lowry method. A pseudokinetic method for the determination of total serum protein employing an initial reading at 3 second (t ) and 10 subsequent readings at 15 second (Δt) intervals has been reported (13). However, this pseudokinetic method was found by other researchers (14) to be relatively imprecise and subject to errors in the analyses of sera from patients with dysproteine- ias. In addition, an instantaneous rate kinetic method for determining total protein in serum has been reported (15, 16). Nevertheless, no one has suggested " the use of the instantaneous rate kinetic method for the determination of total protein in CSF. Summary of the Invention The instant invention encompasses an instan- teneous rate kinetic method for determining the total protein concentration in a CSF sample. In particular, the instantaneous rate kinetic method for determining the total protein concentration in a CSF sample com¬ prises the steps of: (a) reacting the total protein present in the CSF sample wit a biuret reagent to form color reaction products; and (b) εpectrophoto etrically measuring the instantaneous rate of change of the formation of the colored reaction products. The spectrόphotometric measurement is directly propor¬ tional . to the total protein concentration in the CSF sample being assayed. Unlike prior art biuret reaction employed to measure total protein in CSF, the results Obtained via the method of the instant invention are not high in comparison with other procedures such as the preferred turbidimetric technique. In addition, the instantaneous rate kinetic method of the present invention does not require pre- treatment (e.g., centrifugatioh, gel filtration, or precipitation) as suggested by Henry et al. (1). Furthermore, the spectrophotometric measurement can be made at 545 nm because of the increased sensitivity of the instantaneous rate kinetic method of the present invention. - -- — Description of the Preferred Embodiments The instant invention's instantaneous rate kinetic method for determining the total protein con- centration in a CSF sample comprises reacting the total protein in the CSF sample with a biuret reagent to form color reaction products and spectrophotometrically measuring the instantaneous rate of change of the for¬ mation of the color reaction products. The εpectro- photometric measurement, which can be made at about 330 or about 545 nm is directly proportional to the total protein concentration in the sample being assayed. In general, the instantaneous rate of change can be spectrophotometrically measured via any of the well known methods for making such determinations. See, for example, Malmstadt et al., Modern Classics in Analytical Chemistry, 2_:196, Biel ed. , American Chemi¬ cal Society, Washington, D.C. (1976); U.S. Patent 3,933,593; International Congress of Clinical Chemis¬ try, Abstract 1 (1978); and Perry et al., Clinical Chemistry, 23.:6 (1136) (1977). Virtually any biuret reagent having an alka¬ line pH and comprising cupric ions, hydroxyl ions, and a stabilizer can be employed in the instantaneous rate kinetic method of the instant invention. The cupric ions can be present as Cu Y, wherein C Y is any cop- per salt capable of supplying cupric ions. Y, for example, can be selected from a group consisting of S0 4 , Cl 2 , (N0 3 ) 2 , and (Cl0 4 ) 2 . The hydroxyl ions can be present as XOH, wherein X is an alkali metal. Because of their com- mercial availability, sodium hydroxide and potassium hydroxide are the preferred sources of hydroxyl ions.. The alkaline pH of the biuret reagent is preferably greater than 12.5 and more preferably is from about 12.5 to about 13.5. Optimally, the pH can be from about 12.9 to about 13. Virtually any compound which can couple with cupric ions can be employed in the biuret reagent employed in the instant invention. Typical stabilizers include, but are not limited to, tartrate, citrate, and ethylenediaminetetraacetic acid. The biuret reagent can optionally comprise an. agent capable of preventing the auto-oxidation of Cu(OH) 2 . One such compound commonly used in commercial biuret reagents is potassium iodide. The biuret reagent can also optionally con¬ tain a surfactant. Typical surfactants include, but - are not limited to, polyoxyethylene(23) laurylether and octylphenoxy polyethoxy ethanol. Although the concentrations of the various constituents of the biuret reagents employed in the instantaneous rate kinetic method of the instant inven¬ tion are not critical, it is preferred that the sta¬ bilizer be present in an amount sufficient to occupy substantially all of the cupric ion's coordination sites. A preferred biuret reagent for use in con¬ junction with the method of the instant invention com¬ prises CuS0 4 -5H 2 0, NaKC 4 -H 4 0 6 -4H 2 0, KI, NaOH, and C 12 H 25^ OCH 2 CH 2- , 23 OH ( the surfactant being optional ) ; wherein the molar ratio of tartrate to cupric ion is from about 3:1 to about 4:1. More preferably, the molar ratio of tartrate to cupric ion is from about 2.8:1 to about 3.1:1. The following examples are provided for the purpose of further illustration only and are not in- tended to be limitations on the disclosed invention. Example 1 The following. is the composition of a pre¬ ferred biuret reagent for use in conjunction with the instantaneous rate kinetic method for measuring total protein in CSF: Ingredient Concentration CuS0 4 -5H 2 0 8.8 mM/1 NaKC 4 H 4 0 6 -4H 2 0 26.4 mM/1 KI 20 mM/1 NaOH 145 mM/1 C 12 H 25(° CH 2 CH 2 } 23 OH ° *02% w/v pH -13 Example 2 CSF samples (62) were analyzed for total pro- tein via the instantaneous rate kinetic method of the instant invention with the reagent of Example 1 on the ' BURE colorimeter channel of an Astra brand multichannel analyzer (sold by Beckman Instruments, Inc., Fullerton, California). The following parameters were employed in conjunction with this colorimeter: Blanking Time 6.0 seconds Sampling Time 5.5 seconds Total Time 11. 5 seconds Wavelength 546 nm Sample Size 100 μl Reagent Volume 1.0 ml Time Constant of Electronic Circuit 10 seconds Output Voltage ~2.6 volts per change of absorb- ance from 0-100% transmittance The data obtained from this experiment is set forth in Table I. Example 3 The same CSF samples assayed in Example 2 were also assayed via the prior art trichloracetic acid turbidity procedure as set forth in Tietz, Fundamentals of Clinical Chemistry, W. B. Saunders Company, Phila¬ delphia, Penn. (1970) p. 204-206 (17). The data ob¬ tained from this experiment is also set forth in Table I. UR TABLE I Total Protein in CSF Instantaneous Rate Prior Art Kinetic Method of Turbidimetric Sample Present Invention, Y Method, X 1 42 40 2 80 80 3 105 124 4 33 34 5 42 40 6 71 70 7 71 70 8 93 94 9 155 154 10 43 41 11 80 86 12 74 75 13 39 38 14 79 76 15 80 72 16 29 25 17 35 29 18 41 40 19 38 35 20 38 37 21 37 35 22 74 75 23 34 30 24 66 64 25 50 46 26 35 31 27 74 77 28 64 68 29 60 60 30 77 82 31 71 74 32 51 45 33 74 72 34 60 62 35 74 72 36 61 66 37 58 58 38 65 70 39 62 66 40 63 68 41 69 76 42 67 66 43 38 39 44 39 37 45 42 45 46 60 65 47 49 61 48 186 199 49 39 38 50 69 70 51 50 65 52 61 61 53 65 65 54 38 37 55 38 35 56 29 26 57 53 52 58 46 31 10 59 39 38 60 76 78 61 19 16 62 97 96 n = 62 15 Ϋ = 60.4 X = 60.9 Y-Intercept = 5.32 Slope = 0.904 Correlation coefficient = 0.988 The data set forth in Table I indicates that the results obtained via the instantaneous rate kinetic method of the instant invention for determining total protein in CSF are not high, but correlate well, when compared to the results obtained via Henry et al.'s (1) preferred turbidimetric method. This fact is very surprising in view of, inter alia, Henry et al. (1) and Table I of Sethna et al. (3). Based on this disclosure many other modifica¬ tions and ramifications will naturally suggest them- selves to those skilled in the art. These are intended to be comprehended as within the scope of this inven¬ tion. References 1. Henry et al., Clinical Chemistry, Principles and Technics, 2nd Ed., Harper & Row, N.Y., N-Y. (1974) p.422. ' 5 2. Rice, Standard Methods of Clinical Chemistry, Meites ed., Academic Press, N.Y., N.Y. (1965) Vol. 5, p. 231. 3. Sethna et al., Clin. Chem., 3_:249 (1957). 4. ' Dittebrand, Am. J. Clin. Pathol. , 18:439 10 (1948). 5. Goa, Scand. J. Clin. Lab. Invest. , 5_:218 (1953). 6. Kibrick, Clin. Chem., 4:232 (1958). 7. Patrick et al. , Clin. Chem., 9_:283 (1963). 15 8. Rice et al., Clin. Chem., 8_:56 (1962). 9. Rosenthal et al., Clin. Chem., 2_:398 ( " 1956). 10. Ressler et al., Am. J. Clin. Pathol. , 38:131 (1962). 11. Tombs et al. Biochem. J., 73^:13p (1959). 20 12. Watson, Advances in Clinical Chemistry, Sobotka et al. ed. , Academic Press, N.Y., N.Y. (1965) Vol. 8, pp. 237-282 " . 13. Koch et al., Clin. Chem., 20:392 (1974). 14. Savory et al., Clin. Chem., 22_:1102 (1976). 25 15. Perry et al., Clin. Chem., 23_:1136 (1977). 16. Perry, bstracts, International Congress of Clinical Chemistry, Mexico (1978) Abstract #1. 17. Tietz, Fundamentals of Clinical Chemistry, W. B. Saunders Company, Philadelphia, Penn. (1970) p. 30 204-206.

Claims: The embodiments of the invention in which an exclusive property or privilege is claimed are defined as fol¬ lows: 1. A method of determining the total pro- tein concentration in a cerebrospinal fluid sample comprising the steps of: (a) reacting the total protein present in said-cerebrospinal fluid sample with a biuret reagent to form colored reaction products; and (b) spectrophotometrically measuring the instantaneous rate of change of the formation of said colored reaction products; wherein said measurement is directly proportional to said total protein concentration in the sample being assayed. 2. The method of claim 1 wherein said bi¬ uret reagent has an alkaline pH and comprises cupric ions, hydroxyl ions, and a- stabilizer, said stabilizer being present in an amount sufficient to occupy sub- * stantially all coordination sites of said cupric ions. 3. The method of claim 2 wherein said cu- pπc ions are present as Cu Y, wherein Cu is any copper salt capable of supplying cupric ions. 4. The method of claim 2 wherein said hydroxyl ions are present as XOH, wherein X is an alkali metal. 5. The method of claim 2 wherein said alkaline pH is greater than 12.5. 6. The method of claim 2 wherein said alka- line pH is greater than 12.5, said cupric ions are ++ present as Cu Y and said hydroxyl ions are present as XOH, wherein Cu Y is any copper salt capable of sup¬ plying cupric ions and X is an alkali metal. 7. The method of claim 6 wherein said stabilizer is selected from a group consisting of tar¬ trate, citrate, and ethylenediaminetetraacetic acid. 8. The method of claim 6 wherein said alka¬ line pH is from about 12.5 to about 13.5. 9. The method of claim 6 wherein said biur¬ et reagent further comprises an agent capable of pre- venting the auto-oxidation of Cu(OH) 2 . 10. The method of claim 6 wherein said biuret reagent further comprises a surfactant. 11. The method of claim 6 wherein said sta¬ bilizer is selected from a group consisting of tar- trate, citrate, and ethylenediaminetetraacetic acid; said alkaline pH is from about 12.5 to about 13.5; and wherein said biuret reagent further comprises an agent capable of preventing the auto-oxidation of Cu(OH)_ and a surfactant. 12. The method of claim 11 wherein said biuret reagent comprises CuS0 4 « 5H 2 0, NaKC 4 H 4 O g -4H 2 0, KI, NaOH, and C 12 H 25 (OCH 2 CH 2 ) 23 OH; wherein the molar ratio of tartrate to cupric ion is from about 3:1 to about 4:1. 13. The method of claim 12 wherein said biuret reagent has a pH of about 13 and comprises about 8.8 mM/1 CuS0 4 -5H 2 0, about 26.4 mM/1 NaKC 4 H 4 O 6 « 4H 2 0, about 20 mM/1 KI, about 145 mM/1 NaOH, and about 0.02% W V C 12 H 25< 0CH 2 CH 2>23 0H - -

Inventors: JAIN C, SHU F
Applicants: BECKMAN INSTRUMENTS INC
Application year: 1981
Main classification: G01N21
IPC codes: G01N21, G01N33