Clin Biochem. Vol. 24. pp. 3 9 9 - 4 0 6 , 1991
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Purification and Biochemical Characterization of Xanthopterin from Patients with Chronic Renal Failure. I. Isolation, Purification and Preliminary Characterization ROBERT H. WILLIAMS, 1'3 MASHOUF SHAYKH, 2 SAROSH AHMED, 2 THEODORE MUSIALA, 1 NADINE BAZILINSKI, 2'3 GEORGE DUNEA, 2'3 and ALVIN DUBINI1Department of Biochemistry, Rush University and Hektoen Institute for Medical Research, Chicago, IL 60612; 2Division of Nephrology, Cook County Hospital and Hektoen Institute for Medical Research, Chicago, IL 60612; and "~University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA We describe the purification and initial characterization of a hitherto unrecognized fluorescence (excitation/emission maxima at 380/440 nm and 400/460 nm) reported from this laboratory in patients with chronic renal failure (Clin Chem 31: 1988, 1985). Purification was achieved using Sephadex G-10 gel chromatography combined with reverse phase and ion exchange high-performance liquid chromatography (HPLC). Purity of the "blue-green" fluorescent compound was determined to be greater than 99% by HPLC, and two-dimensional thin layer chromatography using an acidic and basic solvent system. The excitation/emission maxima were shown to be 390 nm/456 nm, and ultraviolet scans, at pH 1.0, 7.0, and 13.0, gave absorbance optima at 261 nm/356 nm, 278 nm/ 390 nm, and 255 nm/394 nm, respectively. The isoelectric point of 4.05 in conjunction with the fluorescent and ultraviolet spectra suggests that the fluorophore belongs to the class of compounds known as pteridines.
KEY WORDS: chronic renal failure; uremia; fluorescence; pteridines; high-performance liquid chromatography (HPLC). Introduction
n 1985 we reported a hitherto undescribed fluoImaxima rescence with excitation (Ex):emission (Em) at 380:440 nm and 400:460 nm in the sera of uremic patients (1) that was significantly increased compared to normal subjects (2). This fluorescence was extremely stable to acid/alkaline hydrolysis and was unaffected by treatment with various enzymes (beta-glucuronidase and arylsulfatase), suggesting that it was not an intrinsic property of any peptide or glucuronide/sulfate con-
tDeceased. Correspondence: Robert H. Williams, Ph.D., University of Illinois at Chicago, Department of Pathology -College of Medicine, 1853 West Polk Street, 446 CMW, Chicago, IL 60612, USA. Manuscript received July 7, 1990; revised January 4, 1991 and May 20, 1991; accepted May 23, 1991. CLINICAL BIOCHEMISTRY, VOLUME 24, OCTOBER 1991
jugate (2). The substance contributing to this fluorescence was also readily adsorbed onto charcoal, and then removed by alkalinization to pH 12 (2). Here, we describe our present studies designed to isolate and characterize this compound. Materials a n d m e t h o d s REAGENTS
High-performance liquid chromatography (HPLC) grade triethylamine was obtained from Fisher Scientific Co. (Itasca, IL, USA). HPLC grade acetonitrile, methanol, isopropyl alcohol, butanol, and n-propanol were purchased from Burdick and Jackson (Muskegon, MI, USA). HPLC Grade trifluoroacetic acid and the HPLC internal standard (ndansyl-D-alanyl-glycyl-p-nitro-L-phenylalanyl glycine) was provided by Sigma Chemical Co. (St. Louis, MO, USA). HPLC water was obtained from a Milli-Q Water System purchased from the Millipore Corp. (Milford, MA, USA). All other reagents were A.R. Grade. SAMPLES AND PATIENTS
Predialysis blood samples or hemofiltrate were obtained from 10 patients with chronic renal failure requiring maintenance hemodialysis. Blood was centrifuged, and the separated serum was stored at - 2 0 °C. Hemofiltrate, obtained by standard methods of negative pressure using a dialysis membrane (nominal cut-off 10,000 Da), was stored in the same manner. All subsequent sample analyses were performed at room temperature. ULTRAFILTRATION
Whole serum or hemofiltrate was filtered through a YCO5 "Diaflo" ultra_filtration membrane 399
WILLIAMS, SHAYKH, AHMED, ET AL.
(nominal cut-off of 500 Da) using a Model 8010 Stirred Cell (all from Amicon Corp., Lexington, MA, USA), as described elsewhere (2). The ultrafiltrate was lyophilized and then stored at - 2 0 °C. GEL FILTRATION CHROMATOGRAPHY
The method was modified from Shaykh et al. (1). Serum/hemofiltrate was chromatographed through a 15 mm (i.d.) × 900 mm column of Sephadex G-10 gel, (Pharmacia, Uppsala, Sweden), with a 150 mL bed volume that had been equilibrated with 0.025 M triethylamine acetate at pH 8.0. Absorbance of the effluent was monitored at 254/280 nm simultaneously with a dual-wavelength monitor (Pharmacia, Model 200), using a two-channel recorder (Pharmacia, Model 412) at 10 mV fullscale and a chart speed 25 mm/h. Fractions were collected at 15 min intervals; the flow rate was 20 mL/h. Fractions containing the 380:440/400:460 fluorescence were pooled, lyophilized, and stored at - 20 °C.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
First-stage purification - "reverse p h a s e "
The lyophilized G-10 fractions containing the 380:440/400:460 fluorescence were reconstituted with concentrated triethylamine (TEA) to a pH of 12.0, and the pH was adjusted to 8.0 with acetic acid. Eighteen ~L of the reconstituted G-10 fraction and 2 ~L of internal standard (2 mg/mL) were injected into a Ternary Gradient Liquid Chromatograph (IBM, Model LC/9533, Danbury, CT, USA) fitted with an IBM C~s reverse phase column (5 ~, 4.5 × 250 mm). Mobile phases were 0.025 M TEA acetate, pH 7.78 (Solvent A); 0.025 M TEA acetate in 90% (v/v) acetonitrile, pH 7.78 (Solvent B); and 100% acetonitrile (Solvent C). Gradient elution was obtained with a flow rate of 0.6 mL/min using the following program: isocratic elution with 100% Solvent A for 5 min followed by a gradient elution from 100 to 90% Solvent A and 0 to 10% Solvent B over a 15 min period. The column was regenerated using Solvent C for 15 min, followed by Solvent A for 20 min. The column effluent was monitored using fluorescence detection at Ex 370 nm/Em 418700 nm [IBM, Model (FL) LC9524 Fluorescence Detector] and ultraviolet (UV) detection at 280 nm [IBM, Model (UV) LC9523 Variable UV Wavelength Detector].
"Hydrophase" HP-SAX (10 ~, 7.8 × 100 mm) column (Interaction Chemicals, Inc., Mountain View, CA, USA). Mobile phases were 0.058 M ammonium formate, pH 8.5 (Solvent A) and 0.0565 M formic acid, pH 2.5 (Solvent B). The HPLC program for pH gradient elution was 100% Solvent A for 5 min followed by 100 to 0% Solvent A and 0 to 100% Solvent B over a 30 min period at a flow rate of 2 mL/min. The column was regenerated by pumping Solvent B for 10 min followed by Solvent A for 15 min. Column effluent was monitored using fluorescence and UV detection (as described above). T h i r d - s t a g e purification - "reverse p h a s e "
The method is a modification of that of Niederwieser et al. (3). The lyophilized 380:440/400:460 fraction (from ion exchange HPLC) was reconstituted in 20 ~L of Solvent (A), consisting of HPLC grade isopropyl alcohol, methanol, trifluoroacetic acid, and water in proportions of 5:5:1:990 by volume, respectively, pH adjusted to 2.1. The reconstituted fraction was analyzed by reverse phase HPLC using a Cls (5 ~, 10 × 250 mm) column (III Supplies Co., Meriden, CT, USA). Isocratic elution was performed using 100% Solvent (A) for 15 min at a flow rate of 3 mL/min. The column was regenerated by pumping Solvent (B): isopropyl alcohol, methanol, and trifluoroacetic acid in proportions of 250:250:1 by volume respectively, for 15 min followed by Solvent (A) for 20 min. The column effluent was monitored with fluorescence (Ex 370 nm/Em 418-700 nm) and UV (280, 254, and 210 nm) detection. F o u r t h - s t a g e purification - "reverse p h a s e "
Final purification was achieved by modifying the method of F u k u s h i m a and Nixon (4). The lyophilized 380:440/400:460 fraction (from above) was reconstituted in 20 ~L of Solvent (A) composed of 5% methanol in water (v/v) and analyzed by reverse phase HPLC using a Cls (3 ~, 4.6 × 250 nm) column (Jones Chromatography, Littleton, CO, USA). Isocratic elution was performed with Solvent (A) at a flow rate of 1.2 mL/min for 10 min. The column was regenerated by using Solvent (B) composed of 100% methanol for 15 min followed by Solvent (A) for 20 min. The column effluent was monitored using fluorescence and UV detection (as above).
Second-stage purification - "ion exchange" TWO-DIMENSIONAL THIN LAYERCHROMATOGRAPHY
The lyophilized fraction containing the 380:440/ 400:460 fluorescence (from reverse phase HPLC) was reconstituted in 20 ~L of 0.058 M NH4/OH, pH adjusted to 8.5 with formic acid; both reagents were HPLC Grade. The reconstituted fraction was analyzed by ion exchange HPLC using a polymeric 400
(TLC)
A solution of the purified 380:440/400:460 fraction was made by dissolving 50 ~tg in 0.5 mL of 0.1 M NaOH; 2 ~L of this solution were applied to TLC plates coated with the following adsorbents: Silica Gel G/Silica Gel GF (250 ~, 20 x 20 cm), CLINICAL BIOCHEMISTRY, VOLUME 24, OCTOBER 1991
XANTHOPTERIN IN CHRONIC RENALFAILURE. I MN-300 cellulose/MN-300F cellulose (250 ~, 20 x 20 cm), and high-performance thin layer chromatography Silica Gel HP-HL/Silica HP-HLF (150 ~t, 10 x 10 cm). All plates were obtained from Analtech, Inc. (Newark, DE, USA). The plates were developed in the first dimension by using n-propanol:l% ammonia (2:1) by volume, air dried, and then developed in the second dimension with butanol:acetic acid:water (4:1:1) by volume. Plates were air dried and spots visualized by fluorescence detection using a UV SL-25 Mineralight Lamp at 254 and 366 nm (5,6). ISOELECTRICFOCUSING(IEF) An Ampholine PAG plate, pH range 3.5-9.5 (Pharmacia - LKB, Piscataway, NJ, USA), was cut in half, and then prefocused for 30 min at 10 °C by using a Pharmacia - LKB Multiphor System with the following analytical conditions: voltage = 1500 V, current = 25 mA, power = 15 W. Approximately 250 ~g of the purified 380:440/400:460 fraction was dissolved in 0.1 M sodium hydroxide; 2 ~L of this solution was applied to the cathodic side of the prefocused gel and electrofocused for an additional 30 min. After analysis, the pH gradient across the gel was calibrated by using a Pharmacia - LKB pH surface electrode and the isoelectric point (pI) of the fraction was determined. The fluorescent spot was also visualized for homogeneity. FLUORESCENCEEMISSIONSPECTROSCOPY Fractions collected from gel chromatography and HPLC analysis were monitored m a n u a l l y for fluorescence by using a double monochromator fluorometer (Model 204, Perkin-Elmer Corp., Norwalk, CT). The excitation/emission maxima of each fraction was determined by adding 100 ~L of reconstituted sample to 3.5 mL of 0.025 M triethylamine acetate buffer, pH 8.0. Emission spectra of the fractions obtained from second-stage and fourthstage purification were scanned with a SLM Aminco double monochromator scanning fluorometer (Model SPF-500, SLM Instruments, Inc., Urbana, IL) at excitation wavelengths of 350, 380, and 400 nm, and recorded on a Hewlett Packard graphics plotter. The emission spectra at the peak excitation wavelengths after both stages of HPLC purification were also determined. ULTRAVIOLETSPECTROSCOPY One hundred-eighty jxg of the purified 380:440/ 400:460 fraction was reconstituted in 20 mL of 0.1 M NaOH (pH 13.0), 0.1 M phosphate buffer (pH 7.0), or 0.1 M HC1 (pH 1.0). Approximately 3 mL of each solution with a reagent blank was scanned on a SLM Aminco UV-VIS Double Beam Spectrophotometer (Model Dw-2Rc, SLM Instruments, Inc., Urbana, IL, USA). The UV spectra were reCLINICALBIOCHEMISTRY,VOLUME 24, OCTOBER 1991
corded at an absorbance range of 0.2 (AUFS) and a scan speed of 10 mm/sec. Results A chromatogram representative of the gel chromatography studies is depicted in Figure 1. Each UV fraction was analyzed for fluorescence using a double monochromator fluorometer. The 380:440/ 400:460 fluorophore from ultrafiltrates of serum or hemofiltrate elutes in one distinct UV fraction. A similar pattern was shown to occur for nine other uremic patients. The specific excitation/emission (Ex/Em) maxima of this fraction for each patient sample were: 386/445, 382/442, 380/440, 382/444, 380/438, 382/445, 382/442, 382/444, 382/443, and 382/443. Though some variation is noted, all Ex/Em maxima have spectral characteristics within the range of Ex: 380-386 nm and Em: 438445 nm. Based on the elution position of the molecular weight markers shown in Figure 1, the fluorescent compound has a M r -- 600 Da. By using reverse phase HPLC with a basic solvent system and fluorescence and UV detection, the Sephadex G-10 fraction containing the 380:440/ 400:460 fluorescence was further separated into one prominent fluorescent peak and several UV peaks. The fluorescence chromatogram clearly indicates t h a t the retention behavior was practically identical regardless of the source of the 380:440/ 400:460 fluorophore, i.e., serum or hemofiltrate (Figure 2). It is also apparent from the UV chromatogram (Figure 3) t h a t the compound(s) contributing to the 380:440/400:460 fluorescence constitutes a relatively small percentage of the total UV absorbance from the Sephadex G-10 peak (Figure 1). No fluorescent substances with excitation/emission maxima near the range of Ex: 380-400 nm and Em: 440-460 nm were detected in these other UV fractions or in the pre/post column effluent by m a n u a l analysis with a double monochromator fluorometer. Our previous studies (2) suggested t h a t this fluorescence may be due to an acidic compound. Since m a n y uremic retention products are also acidic and thus tend to be negatively charged at alkaline pH, the fluorescent fraction from reverse phase HPLCwas further purified by ion exchange HPLC by using a strong anion exchanger with a pH gradient system. This separation, using fluorescence and UV detection, is illustrated in Figure 4; only one prominent fluorescent/UV peak is observed. The compound(s) eluted in the pH range of 4 to 5, suggesting t h a t it contained either aromatic/aliphatic (beta or gamma) carboxyl groups or had its isoelectric point near t h a t pH interval. The effluent prior to and after this major fraction was also m a n u a l l y analyzed for fluorescence; again no substances with high excitation/emission maxima were detected. The fluorescent spectral characteristics were determined on this semipurified fraction. The emission peaks at several excitation wavelengths were: Ex 350/Em 450 nm, Ex 380/Em 438 and 401
WILLIAMS, SHAYKH, AHMED, ETAL. r--I
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LEGEND: MOLECULAR WEIGHT (MW) MARKERS DB: DEXTRAN BLUE (MW 2 x 1 0 6 ) DP: DANSYLATED PEPTIDE (MW 629) FA: FOLIC ACID (MW 4 4 1 ) TRP: TRYPTOPHAN (MW 2 0 4 ) UR: URACIL (MW 112)
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