ANALYTICAL
BIOCHEMISTRY
86,
490-497 (1978)
o-Phthalaldehyde for the Fluorometric Nonprotein Amino Compounds DENNIS Bioorganic
J. REEDER,
LORNA
T. SNIEGOSKI,
Assay of
AND ROBERT
SCHAFFER
Standards Section, Analytical Chemistry Division, National Bureau of Standards,’ Washington, D. C. 20234 Received August 23, 1977; accepted December 5. 1977
We describe a manual fluorometric method for the quantitation in protein solutions oftotal free amino compounds, expressed as norleucine. A trichloroacetic acid deproteinization step is employed, and o-phthalaldehyde, buffered with phosphate at pH 9.2, is used as the fluorogenic reagent. The method is linear, reproducible, and rapid. Recoveries of amino acids added to serum are quantitative. Sensitivity is in the picomole range. Results on unselected patient sera are discussed.
During the characterization and certification of a standardized bovine albumin solution as a standard reference material (SRM) for total protein assays, we found a need for a sensitive assay for very low levels of free amino nitrogen compounds that may occur in protein preparations. The method must be able to function in the presence ofthe high concentration of protein which is contained in the SRM. The National Committee for Clinical Laboratory Standards (NCCLS)recommended procedure (1) for this determination of nonprotein amino compounds is based on the ninhydrin technique of Rosen (2). Our initial studies utilizing that method for free amino compounds in the SRM bovine albumin gave poor precision. In an attempt to find a more satisfactory method we tried manual modifications of the automated trinitrobenzenesulfonic acid (TNBS) methodology of Palmer and Peters (3), as personally suggested by Peters. These proved unsatisfactory because precipitation with trichloroacetic acid gave supernatant solutions that were too acidic to be adequately buffered with borax-NaOH for the TNBS reaction. Also, fluorometric methodology (4) with fluorescamine failed to give us adequate precision, as it was too sensitive to differences in pH between controls and samples, and on increasing buffer strength, crystallization of the buffer occurred when I Identification of any commercial product does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose. 0003-2697/78/0862-0490$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
490
FLLJOROMETRIC
ASSAY
491
the fluorescamine in its organic solvent was added to the reaction mixture. However, an alternative fluorometry approach was applicable. The use of o-phthalaldehyde for a-amino acid detection was described initially by Roth (5). The reaction was later modified by Benson and Hare (6) who adapted it for use in an analytical flow system for amino acid analysis. Because of our need to determine free amino compounds accurately at very low concentration levels, we undertook to find optimized reaction conditions. This was achieved by providing better control of pH and use of automatic pipetting. The fluorescence produced is reproducible, linear up to about 100 nmol of norleucine, and sensitive to the 2 to 3 pmol concentration level in serum or albumin at physiological concentrations. On a mass fraction basis, i.e., the mass of amino compound, expressed as norleucine, per unit mass of protein in the solution, a standard deviation of ?O.OOOl can be achieved. MATERIALS
AND METHODS
Reagents. o-Phthalaldehyde was purchased as specially purified crystals for fluorogenic applications (“Fluoropa”) from Durrum Chemical Company, Palo Alto, California 94303. Other grades of o-phthalaldehyde were found to give high blanks and cloudy solutions. Brij 35 was obtained as a 30% solution (w/w in water) from Pierce Chemical Company, Rockford, Illinois. dl-Norleucine was from Sigma Chemical Co., St. Louis, Missouri. Other amino acids and 2-mercaptoethanol were purchased from Calbiochem, San Diego, California. Boric acid was from General Chemicals Division, Allied Chemical Corp. Morristown, New Jersey, and sodium borate was from J. T. Baker Chemical Company, Phillipsburg, New Jersey. All other reagents were analytical grade, used without further purification, and were from Mallinckrodt Chemical Works, St. Louis, Missouri. Fluorometer. Fluorescence measurements were made with a Farrand spectrofluorometer, Catalog No. 117180 (Farrand Optical Company, Inc., Valhalla, New York). The instrument was set manually for excitation and emission wavelengths, using 20-mm slit widths. All measurements were made at room temperature (ca. 22°C). Before each new set of measurements, the instrument was adjusted so that, at an excitation wavelength of 350 nm and an emission wavelength of 450 nm, a standard quinine sulfate solution (250 pg/liter in 0.01 mol/liter H,SO,) in a 1.O-cm cell gave a relative fluorescence of 5.0 on the microammeter. After this initial setting, no further adjustments were made to the gain or zero controls when optimal wavelengths were selected for the assay. The observed readings were then referred to as arbitrary units. Pipetter-diluter. Micromedic automatic pipetters (Model 2500; Micromedic Systems, Inc., Philadelphia. Pennsylvania) were used in both sampling and dispensing modes for measuring reagent solutions.
492
REEDER
ET AL.
Centrifuge. A refrigerated centrifuge (Model J-21 B, Beckman Instruments Co., Inc., Palo Alto, California) equipped with a swinging bucket head (JS 7.5) was used. pH meter. A Model 12 research pH meter (Corning Instruments, Corning, New York) was used for most measurements. For small samples, we used a Beckman Model 1019 (Beckman Instruments, Inc., Fullerton, California) equipped with microelectrodes. Glassware. Glassware was washed thoroughly with detergent, rinsed in tap water and distilled water, then soaked in 3 mol/liter nitric acid for at least 30 min, and finally thoroughly rinsed with distilled water and ovendried. Cleaned glassware was covered with aluminum foil prior to use. Fluorogenic reagent preparation. Phosphate buffer, pH 9.2 was prepared by combining solutions of dibasic sodium phosphate, Na,HPO, (0.43 mol/liter), and tribasic sodium phosphate, Na,PO, (0.36 mol/liter), in a 19: 1 ratio (v/v). The fluorogenic reagent was prepared by dissolving 0.750 g of o-phthalaldehyde in 5 ml of 95% ethanol in a 500-m] volumetric flask, adding 1 ml of 2-mercaptoethanol and 500 ~1 of the Brij solution, and filling to the mark with the phosphate buffer. Sufficient reagent was prepared for a single day’s use. A reagent of related composition, with borate buffer but without Brij 35, was described (7) as being stable in a closed Erlenmeyer flask for 48 hr at room temperature. Torres et al. (8) used a lithium borate buffer at pH 9.2 without Brij 35 in their system and they noted that phosphate buffer gave no interferences in their automated system. Standard stock solutions. A stock norleucine solution of 100 mg/liter was prepared by dissolving microbalance-weighed quantities of norleucine in water. Dilutions for the standard curve were made by using the pipetter-diluter. Stock solutions could be kept 2 to 3 days at 4°C. Deproteinization
and preparation
of working
standards
and blanks.
Deproteinizations were performed by combining (with use of the pipetter-diluter) 4.0 ml of a 6% (w/v) solution of cold (4°C) trichloroacetic acid (TCA) and 1.0 ml of albumin, serum, plasma, water blank, or amino acid standard and mixing thoroughly. Mixtures were allowed to stand for 15 min at 4°C and then centrifuged at 1OOOgfor 15 min at 4°C. The supernatant solutions were used in the test. Assay. By use of the pipetter-diluter, 200 ~1 of TCA-generated supernatant and 3.5 ml of reagent were dispensed into 18 x 125-mm test tubes, in duplicate. After vortex mixing and 30 min of incubation at room temperature, samples were read in the spectrofluorometer. Wavelength maxima for excitation and emission in the Farrand spectrofluorometer were at 351 and 450 nm, respectively, although literature reports generally suggest that 340 nm is to be used as the excitation wavelength and 455 nm as the emission maximum (5,9,10). (The spectrofluorometer was uncorrected for a variety of possible instrumental variations. With this instrument, the
FLUOROMETRIC
ASSAY
493
wavelengths for maximal excitation and emission of quinine sulfate were found to be 352 and 450 nm, respectively, whereas the commonly accepted values are 348 and 452 nm.) RESULTS Reaction
Kinetics
The fluorescence that results after mixing the reagent with the amino acid standard was monitored at 15set intervals for the first 2 min of reaction and at 30-set intervals thereafter. The initial fluorescence observed rose about 25% during the first minute and then declined. The rate of decrease in fluorescence became relatively constant (0.005 arbitrary unitsimin) after 15 min. The fluorescence produced with the deproteinized supernate (or blank) was observed at 5-min intervals for 2 hr. A linear decrease in fluorescence of 0.005 units/min was observed with both after 15 min. Variation
of Fluorescence
with pH
Fluorogenic reagents were prepared as described above, except that 10 buffers with different pH were used. They were prepared by mixing different proportions of 0.43 mol/liter Na2HP0, and NaH,PO, for a pH range of 3.7 to 8.3, with 0.43 mol/liter Na,HPO, and 0.36 mol/liter Na,PO, mixed for pH range of 9.2 to 10.8. Figure 1 illustrates the effects of pH on the fluorescence of blanks and samples containing a fixed amount of norleucine. The fluorescence of the blank is relatively low in the pH range 3.5 to 9.2, but rises rapidly above pH 10, indicating that the test is not as useful above that pH. The maximum in the ratio of fluorescence of norleucine to blank occurs at pH 9.2.
FIG. 1. Effect of pH on the fluorescence of: (A) norleucine (W n ), (B) blank (A A), and (C) ratio of norleucineiblank (x - - - x). Buffers were prepared from 0.43 moliliter Na,HPO, and NaH,PO, or 0.43 mot/liter Na,HPO, and 0.36 mollliter Na,PO,. Norleucine concentration was 2.5 ~mol/liter. The average of duplicate determinations is plotted.
494 Linearity
REEDER
ET AL.
and Sensitivity
In order to minimize analytical variability, a calibration curve was prepared each time the test was run. Quantities of norleucine in the calibration mixtures ranged from 0.05 to 3.24 mg/liter (0.4-24.5 pmol). The standard curve is linear over this range, as shown in Fig. 2, for which the regression equation isy = 0.608x + 0.24, and the correlation coefficient is 0.9996. At the norleucine level of 0.05 mg/liter, readings were approximately three times larger than those of the reagent blank. By adjusting the fluorometer to a more sensitive range and diluting the sample appropriately, we could achieve sensitivity of the assay in the 2 to 3 pmol range. However, with this instrument, signal stability was not adequate for precise determinations at these lower levels of amino compounds. Reactivity
with Amino Acids
The use of Brij-35 with the o-phthalaldehyde reagent was suggested by Schwabe and Catlin (11) and used by Benson and Hare (6) to improve the sensitivity of the reagent to lysine. With Brij-35 in our modified reagent, we found a molar fluorescence ratio of lysine to norleucine of 1.08, and of valine to norleucine of 1.09. However, it should be noted that we have found the molar fluorescence ratio of a glutamic acid-glycine mixture [69:31 (w/w) or approximately equimolar, as described by Klein and Standaert (4)] to norleucine to be 0.66. Analytical
Recovery
Various amounts of a known norleucine solution were added to pooled human serum which had been diluted 1: 10 with 0.15 mol/liter NaCl. The spiked sera were then precipitated with TCA, and duplicate aliquots of the supernates were analyzed. As a second experiment, a glutamic acid-
MICROMOLES
FIG. 2. Linearity of o-phthalaldehyde the average of duplicate determinations. supernatant were used for each level
NOALEUCINE
PER LITER
fluorescence. Typical standard curve. Each point is Optimal assay conditions utilizing 200 ~1 of TCA of norleucine.
FLUOROMETRIC TABLE RECOVERY
Amino
OF KNOWN
LEVELS
acid added b-%)
I
OF AMINO
ACIDS
FROM
HUMAN
SERUM
Total found as norleucine (pg)
Recovery of added (5%)
I.98 2.40 3.05 3.99
100 101 95
2.38 2.67 3.11 3.87
97 99 101
Norleucine None 0.42 1.06 2.1 I Glutamic None 0.30 0.74 1.47
495
ASSAY
acid-glycine
glycine mixture was used at three levels added to diluted human serum. The recoveries obtained in these studies are given in Table 1. Use of Reagent for Long-Term
Studies
Randomly selected samples of the bovine albumin SRM (protein concentration of 70.45 g/liter) were tested for free amino compounds over a period of 4 months. The first of these analyses was performed about 6 months after the purification and preparation of the protein solution. The data are shown in Table 2. Use M’ith Human
Serum and Plasma
Although the major purpose of the development of this method was for use in the assessment of the purity and stability of the albumin SRM, the TABLE FREE
AMINO
COMPOUNDS
AS NORLEUCINE
Storage time at 4°C (months) 0.5 I 2 4 (I Protein concentration h Based on dry weight “fl = 9.
2 IN SRM
BOVINE
Free amino compounds” (mass fraction + I SD)< 0.00023 0.00017 0.00018 0.00023
is 70.45 g/liter. of protein.
ALBUWN”
f t + f
0.00003 0.00001 0.00002 0.00001
496
REEDER
ET AL.
method appeared to be of potential value for clinical use. A random sampling of a daily collection of plasma and serum from a population of patients not known to have aminoacidopathies was obtained from the National Institutes of Health. The mean +-SD of amino nitrogen, expressed as norleucine, in plasma samples was 41 * 2 mg/liter (n = 10). Corresponding results for serum were 37 & 4 mg/liter. Ranges of the determinations were 33 to 47 mg/liter for plasma and 29 to 47 mg/liter for serum. DISCUSSION
The modification introduced here, which employs a different buffer from previously described methods that use o-phthalaldehyde, has the advantage of more adequately controlling the pH of the reaction mixture. The method is a simple and reliable approach to the determination of free amino compounds. It has the advantage of employing a single pipetting step, with one reagent added to a protein-free supernatant. The only limitation to the number of samples assayed at one time would be the number of tubes which could conveniently be dispensed and read in the allotted incubation time. The assay provides adequate sensitivity for the low amino acid levels that may be present in highly purified protein preparations or in normal plasma. o-Phthalaldehyde fluorescence has been reported to decrease rapidly after mixing of reagents (9). For convenience in performing the test as a manual method, but mainly to ensure measurement precision, we found that a 30-min incubation provided a most suitable reaction time. Others, using automated methods, measured fluorescence at times ranging from a few seconds (6,12) to 4 min (8). Although there is a decrease in fluorescence with time, this rate is low (i.e., -0.005 arbitrary fluorescence units/min). The buffer and the ratio of protein supernatant to reagent used may be stabilizing factors under our conditions. The values reported for the total non-protein nitrogen compounds in patient serum or plasma samples are based on a calibration curve which utilized norleucine. Norleucine was used for this work since this was in accordance with the NCCLS method. The values would differ if other amino acids or mixtures of amino acids were used for calibration. Since the sera employed were not representative of a normal, healthy population of donors, the values we report should not be considered as being a normal distribution. They are lower than those of Klein & Standaert (4) who employed the fluorescamine reagent and glycine-glutamic acid for calibration. Higher results were also reported by Goodwin (13) who employed a method which utilizes 2,4-dinitrofluorobenzene. ACKNOWLEDGMENT We are indebted to Dr. Maurice Thanks also to B. Diamondstone
Green for supplying patient serum for initial studies with the ninhydrin
and plasma samples. methodology.
FLUOROMETRIC
ASSAY
497
REFERENCES 1. National Committee for Clinical Laboratory Standards, Standardized Protein Solution (Bovine serum albumin). Approved Standard: ACC-1, October (1972). 2. Rosen, H. (1957) Arch. Biochem. Biophys. 67, lo- 15. 3. Palmer, D. W.. and Peters, T. (1969) C/in. C’11em.15, 891-901. 4. Klein, B., and Standaert, F. (1976) Clirz. Chrm. 22, 413-416. 5. Roth. M. (1971) Anal. Chem. 43, 880-882. 6. Benson, .I. R., and Hare. P. E. (1975) Proc. Nat. Acad. Sci. USA 72, 619-622. 7. Roth, M., and Hampai. A. (1973) J. Chromatogr. 83, 353-356. 8. Torres, A. R.. Alvarez, V. L.. and Sandberg, L. B. (1976) Biochim. Biophys. Acta 434, 209-214. 9. Fourche, J.. Jensen, H., and Neuzil, E. (1976) Anal. Chrm. 48, 155-159. 10. Marton. L. J., and Lee, P. L. Y. (1975) C/in. Chem. 21, 1721-1724. 11. Schwabe, C., and Catlin. J. C. (1974) Anal. Eiochrm. 61, 302-304. 12. Meek, J. L. (1976)Anal. Chem. 48, 375-379. 13. Goodwin, J. F. (1968) Clin. Chem. 14, 1080-1090.
BIOCHEMISTRY
86,
490-497 (1978)
o-Phthalaldehyde for the Fluorometric Nonprotein Amino Compounds DENNIS Bioorganic
J. REEDER,
LORNA
T. SNIEGOSKI,
Assay of
AND ROBERT
SCHAFFER
Standards Section, Analytical Chemistry Division, National Bureau of Standards,’ Washington, D. C. 20234 Received August 23, 1977; accepted December 5. 1977
We describe a manual fluorometric method for the quantitation in protein solutions oftotal free amino compounds, expressed as norleucine. A trichloroacetic acid deproteinization step is employed, and o-phthalaldehyde, buffered with phosphate at pH 9.2, is used as the fluorogenic reagent. The method is linear, reproducible, and rapid. Recoveries of amino acids added to serum are quantitative. Sensitivity is in the picomole range. Results on unselected patient sera are discussed.
During the characterization and certification of a standardized bovine albumin solution as a standard reference material (SRM) for total protein assays, we found a need for a sensitive assay for very low levels of free amino nitrogen compounds that may occur in protein preparations. The method must be able to function in the presence ofthe high concentration of protein which is contained in the SRM. The National Committee for Clinical Laboratory Standards (NCCLS)recommended procedure (1) for this determination of nonprotein amino compounds is based on the ninhydrin technique of Rosen (2). Our initial studies utilizing that method for free amino compounds in the SRM bovine albumin gave poor precision. In an attempt to find a more satisfactory method we tried manual modifications of the automated trinitrobenzenesulfonic acid (TNBS) methodology of Palmer and Peters (3), as personally suggested by Peters. These proved unsatisfactory because precipitation with trichloroacetic acid gave supernatant solutions that were too acidic to be adequately buffered with borax-NaOH for the TNBS reaction. Also, fluorometric methodology (4) with fluorescamine failed to give us adequate precision, as it was too sensitive to differences in pH between controls and samples, and on increasing buffer strength, crystallization of the buffer occurred when I Identification of any commercial product does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose. 0003-2697/78/0862-0490$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
490
FLLJOROMETRIC
ASSAY
491
the fluorescamine in its organic solvent was added to the reaction mixture. However, an alternative fluorometry approach was applicable. The use of o-phthalaldehyde for a-amino acid detection was described initially by Roth (5). The reaction was later modified by Benson and Hare (6) who adapted it for use in an analytical flow system for amino acid analysis. Because of our need to determine free amino compounds accurately at very low concentration levels, we undertook to find optimized reaction conditions. This was achieved by providing better control of pH and use of automatic pipetting. The fluorescence produced is reproducible, linear up to about 100 nmol of norleucine, and sensitive to the 2 to 3 pmol concentration level in serum or albumin at physiological concentrations. On a mass fraction basis, i.e., the mass of amino compound, expressed as norleucine, per unit mass of protein in the solution, a standard deviation of ?O.OOOl can be achieved. MATERIALS
AND METHODS
Reagents. o-Phthalaldehyde was purchased as specially purified crystals for fluorogenic applications (“Fluoropa”) from Durrum Chemical Company, Palo Alto, California 94303. Other grades of o-phthalaldehyde were found to give high blanks and cloudy solutions. Brij 35 was obtained as a 30% solution (w/w in water) from Pierce Chemical Company, Rockford, Illinois. dl-Norleucine was from Sigma Chemical Co., St. Louis, Missouri. Other amino acids and 2-mercaptoethanol were purchased from Calbiochem, San Diego, California. Boric acid was from General Chemicals Division, Allied Chemical Corp. Morristown, New Jersey, and sodium borate was from J. T. Baker Chemical Company, Phillipsburg, New Jersey. All other reagents were analytical grade, used without further purification, and were from Mallinckrodt Chemical Works, St. Louis, Missouri. Fluorometer. Fluorescence measurements were made with a Farrand spectrofluorometer, Catalog No. 117180 (Farrand Optical Company, Inc., Valhalla, New York). The instrument was set manually for excitation and emission wavelengths, using 20-mm slit widths. All measurements were made at room temperature (ca. 22°C). Before each new set of measurements, the instrument was adjusted so that, at an excitation wavelength of 350 nm and an emission wavelength of 450 nm, a standard quinine sulfate solution (250 pg/liter in 0.01 mol/liter H,SO,) in a 1.O-cm cell gave a relative fluorescence of 5.0 on the microammeter. After this initial setting, no further adjustments were made to the gain or zero controls when optimal wavelengths were selected for the assay. The observed readings were then referred to as arbitrary units. Pipetter-diluter. Micromedic automatic pipetters (Model 2500; Micromedic Systems, Inc., Philadelphia. Pennsylvania) were used in both sampling and dispensing modes for measuring reagent solutions.
492
REEDER
ET AL.
Centrifuge. A refrigerated centrifuge (Model J-21 B, Beckman Instruments Co., Inc., Palo Alto, California) equipped with a swinging bucket head (JS 7.5) was used. pH meter. A Model 12 research pH meter (Corning Instruments, Corning, New York) was used for most measurements. For small samples, we used a Beckman Model 1019 (Beckman Instruments, Inc., Fullerton, California) equipped with microelectrodes. Glassware. Glassware was washed thoroughly with detergent, rinsed in tap water and distilled water, then soaked in 3 mol/liter nitric acid for at least 30 min, and finally thoroughly rinsed with distilled water and ovendried. Cleaned glassware was covered with aluminum foil prior to use. Fluorogenic reagent preparation. Phosphate buffer, pH 9.2 was prepared by combining solutions of dibasic sodium phosphate, Na,HPO, (0.43 mol/liter), and tribasic sodium phosphate, Na,PO, (0.36 mol/liter), in a 19: 1 ratio (v/v). The fluorogenic reagent was prepared by dissolving 0.750 g of o-phthalaldehyde in 5 ml of 95% ethanol in a 500-m] volumetric flask, adding 1 ml of 2-mercaptoethanol and 500 ~1 of the Brij solution, and filling to the mark with the phosphate buffer. Sufficient reagent was prepared for a single day’s use. A reagent of related composition, with borate buffer but without Brij 35, was described (7) as being stable in a closed Erlenmeyer flask for 48 hr at room temperature. Torres et al. (8) used a lithium borate buffer at pH 9.2 without Brij 35 in their system and they noted that phosphate buffer gave no interferences in their automated system. Standard stock solutions. A stock norleucine solution of 100 mg/liter was prepared by dissolving microbalance-weighed quantities of norleucine in water. Dilutions for the standard curve were made by using the pipetter-diluter. Stock solutions could be kept 2 to 3 days at 4°C. Deproteinization
and preparation
of working
standards
and blanks.
Deproteinizations were performed by combining (with use of the pipetter-diluter) 4.0 ml of a 6% (w/v) solution of cold (4°C) trichloroacetic acid (TCA) and 1.0 ml of albumin, serum, plasma, water blank, or amino acid standard and mixing thoroughly. Mixtures were allowed to stand for 15 min at 4°C and then centrifuged at 1OOOgfor 15 min at 4°C. The supernatant solutions were used in the test. Assay. By use of the pipetter-diluter, 200 ~1 of TCA-generated supernatant and 3.5 ml of reagent were dispensed into 18 x 125-mm test tubes, in duplicate. After vortex mixing and 30 min of incubation at room temperature, samples were read in the spectrofluorometer. Wavelength maxima for excitation and emission in the Farrand spectrofluorometer were at 351 and 450 nm, respectively, although literature reports generally suggest that 340 nm is to be used as the excitation wavelength and 455 nm as the emission maximum (5,9,10). (The spectrofluorometer was uncorrected for a variety of possible instrumental variations. With this instrument, the
FLUOROMETRIC
ASSAY
493
wavelengths for maximal excitation and emission of quinine sulfate were found to be 352 and 450 nm, respectively, whereas the commonly accepted values are 348 and 452 nm.) RESULTS Reaction
Kinetics
The fluorescence that results after mixing the reagent with the amino acid standard was monitored at 15set intervals for the first 2 min of reaction and at 30-set intervals thereafter. The initial fluorescence observed rose about 25% during the first minute and then declined. The rate of decrease in fluorescence became relatively constant (0.005 arbitrary unitsimin) after 15 min. The fluorescence produced with the deproteinized supernate (or blank) was observed at 5-min intervals for 2 hr. A linear decrease in fluorescence of 0.005 units/min was observed with both after 15 min. Variation
of Fluorescence
with pH
Fluorogenic reagents were prepared as described above, except that 10 buffers with different pH were used. They were prepared by mixing different proportions of 0.43 mol/liter Na2HP0, and NaH,PO, for a pH range of 3.7 to 8.3, with 0.43 mol/liter Na,HPO, and 0.36 mol/liter Na,PO, mixed for pH range of 9.2 to 10.8. Figure 1 illustrates the effects of pH on the fluorescence of blanks and samples containing a fixed amount of norleucine. The fluorescence of the blank is relatively low in the pH range 3.5 to 9.2, but rises rapidly above pH 10, indicating that the test is not as useful above that pH. The maximum in the ratio of fluorescence of norleucine to blank occurs at pH 9.2.
FIG. 1. Effect of pH on the fluorescence of: (A) norleucine (W n ), (B) blank (A A), and (C) ratio of norleucineiblank (x - - - x). Buffers were prepared from 0.43 moliliter Na,HPO, and NaH,PO, or 0.43 mot/liter Na,HPO, and 0.36 mollliter Na,PO,. Norleucine concentration was 2.5 ~mol/liter. The average of duplicate determinations is plotted.
494 Linearity
REEDER
ET AL.
and Sensitivity
In order to minimize analytical variability, a calibration curve was prepared each time the test was run. Quantities of norleucine in the calibration mixtures ranged from 0.05 to 3.24 mg/liter (0.4-24.5 pmol). The standard curve is linear over this range, as shown in Fig. 2, for which the regression equation isy = 0.608x + 0.24, and the correlation coefficient is 0.9996. At the norleucine level of 0.05 mg/liter, readings were approximately three times larger than those of the reagent blank. By adjusting the fluorometer to a more sensitive range and diluting the sample appropriately, we could achieve sensitivity of the assay in the 2 to 3 pmol range. However, with this instrument, signal stability was not adequate for precise determinations at these lower levels of amino compounds. Reactivity
with Amino Acids
The use of Brij-35 with the o-phthalaldehyde reagent was suggested by Schwabe and Catlin (11) and used by Benson and Hare (6) to improve the sensitivity of the reagent to lysine. With Brij-35 in our modified reagent, we found a molar fluorescence ratio of lysine to norleucine of 1.08, and of valine to norleucine of 1.09. However, it should be noted that we have found the molar fluorescence ratio of a glutamic acid-glycine mixture [69:31 (w/w) or approximately equimolar, as described by Klein and Standaert (4)] to norleucine to be 0.66. Analytical
Recovery
Various amounts of a known norleucine solution were added to pooled human serum which had been diluted 1: 10 with 0.15 mol/liter NaCl. The spiked sera were then precipitated with TCA, and duplicate aliquots of the supernates were analyzed. As a second experiment, a glutamic acid-
MICROMOLES
FIG. 2. Linearity of o-phthalaldehyde the average of duplicate determinations. supernatant were used for each level
NOALEUCINE
PER LITER
fluorescence. Typical standard curve. Each point is Optimal assay conditions utilizing 200 ~1 of TCA of norleucine.
FLUOROMETRIC TABLE RECOVERY
Amino
OF KNOWN
LEVELS
acid added b-%)
I
OF AMINO
ACIDS
FROM
HUMAN
SERUM
Total found as norleucine (pg)
Recovery of added (5%)
I.98 2.40 3.05 3.99
100 101 95
2.38 2.67 3.11 3.87
97 99 101
Norleucine None 0.42 1.06 2.1 I Glutamic None 0.30 0.74 1.47
495
ASSAY
acid-glycine
glycine mixture was used at three levels added to diluted human serum. The recoveries obtained in these studies are given in Table 1. Use of Reagent for Long-Term
Studies
Randomly selected samples of the bovine albumin SRM (protein concentration of 70.45 g/liter) were tested for free amino compounds over a period of 4 months. The first of these analyses was performed about 6 months after the purification and preparation of the protein solution. The data are shown in Table 2. Use M’ith Human
Serum and Plasma
Although the major purpose of the development of this method was for use in the assessment of the purity and stability of the albumin SRM, the TABLE FREE
AMINO
COMPOUNDS
AS NORLEUCINE
Storage time at 4°C (months) 0.5 I 2 4 (I Protein concentration h Based on dry weight “fl = 9.
2 IN SRM
BOVINE
Free amino compounds” (mass fraction + I SD)< 0.00023 0.00017 0.00018 0.00023
is 70.45 g/liter. of protein.
ALBUWN”
f t + f
0.00003 0.00001 0.00002 0.00001
496
REEDER
ET AL.
method appeared to be of potential value for clinical use. A random sampling of a daily collection of plasma and serum from a population of patients not known to have aminoacidopathies was obtained from the National Institutes of Health. The mean +-SD of amino nitrogen, expressed as norleucine, in plasma samples was 41 * 2 mg/liter (n = 10). Corresponding results for serum were 37 & 4 mg/liter. Ranges of the determinations were 33 to 47 mg/liter for plasma and 29 to 47 mg/liter for serum. DISCUSSION
The modification introduced here, which employs a different buffer from previously described methods that use o-phthalaldehyde, has the advantage of more adequately controlling the pH of the reaction mixture. The method is a simple and reliable approach to the determination of free amino compounds. It has the advantage of employing a single pipetting step, with one reagent added to a protein-free supernatant. The only limitation to the number of samples assayed at one time would be the number of tubes which could conveniently be dispensed and read in the allotted incubation time. The assay provides adequate sensitivity for the low amino acid levels that may be present in highly purified protein preparations or in normal plasma. o-Phthalaldehyde fluorescence has been reported to decrease rapidly after mixing of reagents (9). For convenience in performing the test as a manual method, but mainly to ensure measurement precision, we found that a 30-min incubation provided a most suitable reaction time. Others, using automated methods, measured fluorescence at times ranging from a few seconds (6,12) to 4 min (8). Although there is a decrease in fluorescence with time, this rate is low (i.e., -0.005 arbitrary fluorescence units/min). The buffer and the ratio of protein supernatant to reagent used may be stabilizing factors under our conditions. The values reported for the total non-protein nitrogen compounds in patient serum or plasma samples are based on a calibration curve which utilized norleucine. Norleucine was used for this work since this was in accordance with the NCCLS method. The values would differ if other amino acids or mixtures of amino acids were used for calibration. Since the sera employed were not representative of a normal, healthy population of donors, the values we report should not be considered as being a normal distribution. They are lower than those of Klein & Standaert (4) who employed the fluorescamine reagent and glycine-glutamic acid for calibration. Higher results were also reported by Goodwin (13) who employed a method which utilizes 2,4-dinitrofluorobenzene. ACKNOWLEDGMENT We are indebted to Dr. Maurice Thanks also to B. Diamondstone
Green for supplying patient serum for initial studies with the ninhydrin
and plasma samples. methodology.
FLUOROMETRIC
ASSAY
497
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