ANALYTICAL

BIOCHEMISTRY

Automated

GEORGE

Department

84,

231-239 (1978)

Fluorometric Determination of Free and Total Tryptophan in Plasma A. MASON,’

JAMES A. DIEZ,~ HARRY H. DUTTON, AND GEORGE K. SUMMER

of Biochemistry and Nutrition, University MacNider Building-202H, Chapel Hill,

of North Carolina at Chapel North Carolina 27514

Hill,

Received June 13, 1977; accepted September 8, 1977 We have developed a sensitive automated fluorometric method based upon the manual procedure of Denckla and Dewey [(1967) J. Lab. Clin. Med. 69, 160-1691 for determining both free and total tryptophan in plasma. Free tryptophan is measured in a series of increasingly diluted aliquots of a sample of plasma after tryptophan bound to albumin is removed by continuous-flow dialysis. Free and total tryptophan are then derived from Scatchard plots of the data. The method can be used for nutritional assessments, clinical investigation of behavioral disorders in which serotonin is implicated in the pathogenesis, and studies on tryptophan transport and metabolism.

Tryptophan is unique among naturally occurring amino acids in that it occurs in plasma both free in solution and bound to albumin (1). Although free tryptophan in plasma is generally considered to represent the pool available for transport into other tissues (2-4), total plasma tryptophan levels sometimes appear to correlate better with levels in other tissues (5,6). Tryptophan is the precursor of serotonin (Shydroxytryptamine), a neurotransmitter implicated in the regulation of a variety of behaviors. Recent interest in levels of free and bound tryptophan in plasma stems from suggestions that these forms of the amino acid may result from or contribute to abnormalities in the metabolism of serotonin and, in turn, be causally related to depression, hyperactivity, and other affective disorders (7,B). Tryptophan in plasma is most often quantified by the manual fluorometric method of Denckla and Dewey (9). In this procedure, the fluorophore, norharmon, is formed from tryptophan by condensation with formaldehyde followed by oxidation with FeCl, in trichloroacetic acid (TCA) and HCl at 100°C. for 1 hr. Total tryptophan in plasma is ’ To whom correspondence should be addressed. * Present address: School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332. 231

0003-2697/78/0841-0231$02.00/O Copyright All rights

0 1978 by Academic Press. Inc. of reproduction in any form reserved.

232

MASON

ETAL.

measured after tryptophan bound to albumin is released by treatment of the sample with TCA to precipitate protein. Several procedures for determining free tryptophan in plasma have been reported (1,4,10). Most workers prefer to measure free tryptophan in ultrafiltrates of plasma; however, such methods are generally very time consuming, and because of the lability of the equilibrium between free and bound plasma tryptophan, estimates of free tryptophan by such techniques may vary considerably (5). We have developed an automated fluorometric method for measuring both free and total tryptophan. Although the chemistry of the method described herein is basically the same as that of the manual method of Denckla and Dewey (9), several recently reported modifications (10,ll) have been incorporated into our procedure which improve accuracy and allow adaptation to automation. MATERIALS

AND METHODS

Preparation of samples. Approximately 4 ml of whole blood was collected by venipuncture using Venoject vacuum blood-collection tubes (Kimble-Terumo) containing sodium heparin (1.43 III/ml) as an anticoagulant. Plasma was collected after centrifugation of the samples at 2200g for 5 min at room temperature. All samples were analyzed within 24 hr of collection. Reagents for automated method. Stock phosphate buffer: A 0.1 M phosphate buffer (pH 7.35) is prepared in distilled water. Phosphate-buffered saline (PBS): NaCl, 0.85%, in 0.01 M phosphate buffer (pH 7.35) containing 1 ml/1000 ml of Brij 35 (Fisher), a surfactant composed of polyoxyethylene ethers of fatty alcohols. The Brij 35 is omitted if the PBS is to be used in preparing tryptophan standards or diluting plasma. FeCl, Reagent: A 3.3 x 10v3 M solution of FeCl, in 12% perchloric acid containing 1 ml of Brij/lOOO ml. This reagent is stable for several days. Formaldehyde: A freshly prepared 7.2% (v/v) aqueous solution of formaldehyde with 1 ml of Brij/lOOO ml. Tryptophan standards: A stock 100 microgram per milliliter solution of tryptophan in PBS is diluted with PBS to obtain tryptophan standards of the desired concentration (0.01 to 5.0 pg/ml). Automated method. The manifold shown in Fig. 1 consists of AutoAnalyzer modules (Technicon). Samples are taken up by a glass sampler needle and immediately dialyzed against PBS for removal of protein. The dialysate containing free tryptophan is first mixed with formaldehyde and then with the FeCl, reagent, and is passed through a 40-ft glass coil heated to 96°C in a circulating oil bath. After cooling, the fluorescence of norharmon is measured in a fluoronephelometer (Technicon) with Kodak Wratten 18A primary and 2A secondary filters.

AUTOMATED

DETERMINATION

TRYPTOPHAN

OF TRYPTOPHAN

233

MANIFOLD SAMPLER II

FLUOROMETER

FIG.

RECORDER

1. Flow diagram for automated fluorometric determination

of tryptophan.

A glass sampler needle rather than a stainless steel needle is used to eliminate the possibility of molybdenum contamination which Denckla and Dewey (9) report can inhibit the reaction. Perchloric acid is preferred over TCA for preparing the FeCl, reagent because TCA, when heated in the glass coils, produces CO, which causes an erratic flow rate and increased sample carryover in the automated system. The FeCl, reagent is added after the formaldehyde and just before heating to improve the accuracy of the determination (11). This also prevents exposure of the reaction mixture to light for more than 2 min after the ferric chloride reagent is added at which time the mixture is sensitive to uv light (12). If the manifold is to be exposed to a source of uv light the system should be shielded from the point at which the ferric chloride reagent is added (the second mixing coil) to the oil bath. A sampling rate of 40/hr with a sample-to-wash ratio of 1:2 is used for analysis of tryptophan standards, deprotenized plasma, or plasma ultrafiltrates. Whole or diluted plasma is sampled at a rate of 30/hr with a sample-to-wash ratio of 1: 13. This unconventional sample-to-wash ratio is achieved with a Model 4016 Sampler-Timer (Gilford): the sample rate is set at 60/hr, the sample-to-wash ratio is set at 1:6, and a wash cup containing PBS is placed between samples. The lower sampling rate and longer wash are needed to prevent carryover between the more viscous plasma samples.. Determination

of free and total tryptophan from Scatchard plots.

Studies of tryptophan binding to serum albumin have demonstrated that dissociation, facilitated by dilution, follows first-order kinetics (1). Therefore, Scatchard plots (13) of tryptophan binding data should be straight

234

MASON

ET AL.

lines, and theoretically one should be able to derive the concentrations of both free and total tryptophan from these data. To test this hypothesis, portions of fresh plasma samples were diluted from 1.2 to 30 times with PBS, and the concentration of free tryptophan of each diluted portion was then determined by the automated method. Scatchard plots (micrograms per milliliter of tryptophan versus micrograms per milliliter of tryptophan per dilution factor) were prepared from these data. The concentration of total tryptophan was obtained by extrapolation of the leastsquares line to infinite dilution (the x intercept), and the concentration of free tryptophan was taken as that point on the least-squares line where x = y (when the dilution factor is 1). Both values were derived by a PDP-8/L computer (Digital Equipment Corp.) or a SR5 1A pocket calculator (Texas Instruments) using a modified least-squares program. Tyrosine, which is unbound in plasma, was also determined in these same diluted portions by an automated method (14) which uses continuous-flow dialysis for removal of protein. Scatchard plots of the tyrosine data were compared to the tryptophan results to determine if apparent increases in concentration of free tryptophan produced by dilution of plasma were accentuated by dialysis. Comparative analytical studies. Plasma ultrafiltrates for determination of free tryptophan, were prepared using CF 50 A Centriflo membrane cones (Amicon). Centrifugation at 1OOOgwas performed at 35°C for 1 hr. In a later experiment, ultrafiltrates were collected at IO-, 20-, 30-, and 40-min intervals during centrifugation. Whole plasma was diluted I:30 with 12% TCA for precipitation of protein. The concentration of tryptophan in ultrafiltrates and the acid-precipitated plasmas were measured by the manual method of Bloxam and Warren (ll), a modification of the Denckla and Dewey method (9), and by the automated method. Recovery of tryptophan from plasma. Different amounts of tryptophan (2-10 pg/ml) were added in 100 ~1 of PBS to l-ml portions of plasma. These plasma samples were then diluted with PBS to obtain final dilutions of 1:2.2, 1:4.4, and 1:8.8. The free tryptophan concentrations of these diluted plasmas, measured by the automated method, were used to construct Scatchard plots from which estimates of total tryptophan were obtained. RESULTS AND DISCUSSION

For measuring the concentration of tryptophan in standards, deproteinized plasma, or plasma ultrafiltrates, the automated method can be operated continuously at a rate of 40 samples/hr, with excellent reproducibility and negligible carryover between adjacent samples (Fig. 2). Modification of the sampling rate and sample-to-wash ratio, as described, is necessary to achieve comparable results with whole or diluted plasma.

AUTOMATED

DETERMINATION

OF TRYPTOPHAN

235

FIG. 2. Typical recorder tracing of tryptophan standards at 40 samples/hr with a sampleto-wash ratio of I:2 showing (left to right) steady-state (0.6 &ml), reproducibility (4 x 0.6~&ml standards), carryover (0.2-, l.O-, 0.2~&ml standards), and tryptophan standards (0.2, 0.2, 0.4, 0.4, 0.6, 0.6, 0.8, 0.8, 1.0 pg/ml, and the reverse).

The automated method proved to be equivalent in sensitivity and accuracy to the manual procedures to which it was compared. Although somewhat better reproducibility of determinations was obtained using the automated method, its greatest advantage is superior speed, particularly in measuring the concentration of free tryptophan in plasma samples. In Table 1, values for free and total tryptophan determined directly by the automated method from respective analyses of undiluted plasma and plasma diluted 1:30 with 12% TCA are compared to values derived from Scatchard plots. There is excellent agreement between the values for free tryptophan by the two determinations (r = 0.997). The values for total tryptophan derived from Scatchard plots are somewhat higher than those obtained from analysis of acid-precipitated plasma, but the correlation of the results obtained by the two strategies is excellent (r = 0.993). As shown in Table 2, recoveries of total tryptophan from plasma averaged 100.6%. The validity of the automated method is dependent upon quantitative dialysis of free tryptophan in solutions of plasma containing variable concentrations of tryptophan and protein. The effect of diluting plasma on the concentration of free tryptophan and free tyrosine (an unbound amino acid in plasma) is shown in Fig. 3. The tryptophan binding curve has the hyperbolic shape characteristic of the saturation phenomenon of enzymecatalyzed reactions. At low (physiological) concentrations of tryptophan, the proportion of tryptophan that is free varies inversely with tryptophan concentration. At dilutions of 1:20 or more virtually all of the tryptophan is free. In contrast, the tyrosine binding curve is a horizontal line indicating that all of the tyrosine is free. This demonstrates that continuous-flow dialysis of tyrosine in plasma of different dilutions is quantitative; it is likely that this is also true of tryptophan. The slope of

236

MASON ET AL. TABLE

1

COMPARISONOFVALUESFORFREEANDTOTALTRY~TOPHANIN

PLASMA'

Automated method Direct Plasma sample

Free (mhnl)

Total b-dml)

FC FK JD ST 507 156 510 536 538 568 G.P. BB

3.01 3.15 2.97 2.32 5.14 5.05 3.18 3.01 2.62 3.58 7.32 5.93

13.00 17.37 11.82 11.99 7.17 12.35 5.62 5.84 4.51 8.46 13.60 12.69

Graphic Percentage free

Free (w&W

Total b.&-nl)

Percentage free

23 18 25 19 72 41 57 52 58 42 54 47

2.98 3.21 3.00 2.50 5.19 4.39 3.13 2.90 2.67 3.53 7.29 6.17

15.13 20.34 14.28 14.05 7.64 13.63 5.97 5.98 4.91 9.03 14.49 13.44

20 16 21 18 68 36 52 48 54 39 50 46

a Values for free and total tryptophan determined directly respective analyses of undiluted plasma and plasma diluted pared to values derived from Scatchard plots. By the two r = 0.997, slope = 1.000, intercept = 0.013; and for total = 1.194, intercept = -0.801.

by the automated method from 1:30 with 12% TCA are commethods, for free tryptophan: tryptophan: r = 0.993, slope

Scatchard plots of tryptophan binding data increases with the percentage of free tryptophan (Fig. 4). The Scatchard plot of the tyrosine data from Fig. 3 is a vertical line. Values of free tryptophan in plasma ultrafiltrates prepared using Centriflo membrane cones were consistently lower and more variable TABLE RECOVERYOFTOTALTRYPTOPHAN

2 FROM

PLASMA

Tryptophan (pg/mt)

n Mean.

Expected

Found

16.78 18.78 20.78 22.78 24.78

14.78 16.75 18.49 21.04 22.87 25.61

Recovery (%) 96.5 92.8 104.3 101.1 108.3 100.6”

AUTOMATED

0

DETERMINATION

5

10

15

DILUTION

20

OF TRYPTOPHAN

25

237

30

FACTOR

FIG. 3. Comparison of the levels of free tryptophan (TRP) and tyrosine (TYR) determined in increasingly diluted (1.2-30 times) aliquots of a sample of plasma, demonstrating the effect of dilution on the binding of tryptophan by albumin.

than those obtained using the automated method. In addition, we were unable to construct linear Scatchard plots from the values of free tryptophan in ultrafiltrates from series of increasingly dilute plasma. Both of these findings appear to be related to the influence of the tryptophan:albumin ratio on tryptophan binding. As the ratio of albumin (available tryptophan binding sites) to free tryptophan in the Centriflow cones increases during ultrafiltration, the apparent concentration of free tryptophan decreases (Table 3). The validity of the results obtained

FIG. 4. Scatchard plots of tryptophan binding data from samples of plasma from subjects (CW, FC, 507, 510) with different percentages of free tryptophan.

238

MASON ET AL. TABLE

3

EFFECTOFCENTRIFUGATIONTIMEONTHEAPPARENTCONCENTRATION OFFREETRYPTOPHAN IN PLASMA Tryptophan (&ml) Centriflo cone method Sample

Automated method

10 mitt”

20 min

30 min

40 min

1 2

3.21 7.29

2.93 5.05

2.24 3.37

1.88 3.20

1.72 2.05

a Time of filtrate collection.

using the automated method is demonstrated by the linearity of Scatchard plots of binding data from samples of plasma with different percentages of free tryptophan (Fig. 4). The brief continous-flow dialysis apparently does not produce errors in determining free tryptophan as significant as those associated with the protein-concentrating effect of the Centriflo cone method. Furthermore, the use of Scatchard plots permits the measurement of free tryptophan in samples which require dilution, such as tissue homogenates. Free and total tryptophan can be determined in large numbers of plasma samples using the automated method by measuring free tryptophan in three (or more) dilutions of each sample, e.g., 1:2, 1:4, and 1:8, and deriving both free and total tryptophan from Scatchard plots. An alternate procedure would be to measure an undiluted sample for free tryptophan and a 1:30 dilution (with 12% TCA or PBS) for total tryptophan. Using either procedure both free and total tryptophan can be determined in less than one milliliter of plasma. The method should therefore be suitable for nutritional evaluation of patients, clinical studies on behavioral disorders in which the availability of tryptophan, as the precursor of the neurotransmitter serotonin, may be involved, and research on tryptophan transport and metabolism. ACKNOWLEDGMENTS Supported by Project No. 236, Bureau of Community Health Services, Health Services Administration, DHEW, USPHS Grants HD 03110 and RR-46, American Cancer Society Grant No. IN-ISQ, and a grant from the North Carolina United Way, Inc.

REFERENCES 1. McMenamy,

C. C., Van Mercke, J., and Onclay, J. L. (1961) 135-139. 2. Tagliamonte, A., Biggio, G., and Gessa, G. L. (1971) Rev. Ter. 11, 251-255. 3. Knott, P. J., and Curzon, G. (1972) Nature (London) 239, 452-453. Arch.

R. H., Lund,

Biochem.

Biophys.

93,

AUTOMATED 4. Tagliamonte,

DETERMINATION

A., Biggio, G., Vargiu,

OF TRYPTOPHAN

239

L., and Gessa, L. G. (1973) Life Sci. 12,

217-287.

5. Madras, B. K., Cohen, E. L., Messing, R., Munro, H. N., and Wurtman, R. J. (1974) Metabolism 23, 1107-1116. 6. Pardridge, W. M. (1977)J. Neurochem. 28, 103-108. 7. Coppen, A., Brooksband, B. W. L., Eccleston, E., Peet, M., and White, S. G. (1974) Psychol. Med. 4, 164-173. 8. Brase, D. A., and Loh, H. H. (1975) Life Sci. 16, 1005-1016. 9. Denckla, W. D., and Dewey, H. K. (1967) J. Lab. Clin. Med. 69, 160-169. 10. Eccleston, E. G. (1973) Clin. Chim. Acra 48, 269-272. 11. Bloxam, D. L., and Warren, W. H. (1974) Anal. Biochem. 60, 621-625. 12. Lehmann, J. (1971) Sand. J. Clin. Lob. Invest. 28, 49-55. 13. Scatchard, G. (1949) Ann. N.Y. Acad. Sci 51, 660-669. 14. Blau, K., and Edwards, D. J. (1971) Biochem. Med. 5, 333-341.

Automated fluorometric determination of free and total tryptophan in plasma.

ANALYTICAL BIOCHEMISTRY Automated GEORGE Department 84, 231-239 (1978) Fluorometric Determination of Free and Total Tryptophan in Plasma A. MAS...
478KB Sizes 0 Downloads 0 Views