Clin Biochem. Vol. 24. pp. 407--415, 1991 Printed in Canada. All rights reserved.
0009-9120/91 $3.00 + .00
Cbpyright c 1991 The Canadian Society of Clinical Chemists.
Purification and Biochemical Characterization of Xanthopterin from Patients with Chronic Renal Failure. I1. Biochemical Elucidation and Structural Analysis ROBERT H. WILLIAMS, 1'3 MASHOUF SHAYKH, 2 SAROSH AHMED, 2 THEODORE MUSIALA, 1 NADINE BAZILINSKI, 2'3 GEORGE DUNEA, 2'3 and ALVIN DUBINI 1Department 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 have identified the primary endogenous fluorescent substance, which has characteristic excitation/emission maxima at 380/440 nm and 400/460 nm, found in the sera of patients with chronic renal failure (Clin Chem 32: 1276, 1988). Preliminary studies, using thin layer chromatography (with cellulose) in conjunction with pteridine standards, indicated that the compound is an unconjugated pteridine. Characterization by gas chromatography-mass spectrometry (electron impact), direct probe-mass spectrometry (electron impact/chemical ionization), and Fourier Transform Infrared analysis showed this compound to be xanthopterin (2-amino 4,6 pteridinedione), an unconjugated pteridine known to be present in man in trace quantities. An authentic sample of this compound had a retention time with high-performance liquid chromatography (HPLC) identical to that of the purified fluorophore. The physiological role of xanthopterin in the pathogenesis of uremia has yet to be elucidated.
KEY WORDS: chronic renal failure; uremia; pteridine; xanthopterin; fluorescence; gas chromatographymass spectrometry; infrared analysis. Introduction recent years, several investigators have found Ithenvarious endogenous fluorescent compounds in sera of patients with chronic renal failure. Some of these represent tryptophan metabolites or derivatives; others have not yet been characterized (1-3). During our earlier studies of uremic retention products, the so-called middle molecules (4-6), we found a hitherto unreported fluorescent compound and described its purification and initial characterization (7). Here, using the purified mate-
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
rial obtained from our previous work, we describe the studies used to elucidate its structure. Methods and materials REAGENTS
Thioglycolic acid, peptides [alpha melanocyte stimulating hormone (alpha MSH), and oxytocin] and pteridine standards [6-hydroxymethylpterin, 6-methylpterin, xanthopterin (dione), leucopterin, pteroic acid, folic acid] were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Other pteridine standards [6-biopterin, isoxanthopterin, lumazine, L-monapterin, D(+)-neopterin, pterin, pterin-6-caraboxylic acid, xanthopterin monohydrate] were bought from the Fluka Chemical Corp. (Ronkonkoma, NY, USA). High-performance liquid chromatography (HPLC) grade acetonitrile, n-propanol, butanol, isopropanol, and ethanol were obtained from Burdick and Jackson (Muskegon, MI, USA), HPLC grade triethylamine from Fisher Scientific (Itasca, IL, USA), A.C.S. grade phenol from Aldrich Chemical Co. (Milwaukee, WI, USA), and 6 N constant boiling HC1 and N, O-BiB (trimethylsilyl) trifluoracetamide (BSTFA) from Pierce Chemical Co. (Rockford, IL, USA). BLOOD SAMPLESAND PREPARATION
Predialysis blood samples were obtained from 79 patients with chronic renal failure (CRF) requiring maintenance hemodialysis; none had taken medication that day. Blood was centrifuged, and the separated serum was stored at - 2 0 °C until analyzed. Control sera from 34 normal subjects were obtained in a similar manner. Whole serum was filtered through a YCO5 "Diaflo" ultrafiltration
407
WILLIAMS, SHAYKH, AHMED, ET AL.
membrane (nominal cut off 500 Da) by using a Model 8010 Stirred Cell (all from Amicon Corp., Lexington, MA, USA), as described elsewhere (5). THIN LAYER CHROMATOGRAPHY STANDARDS
(TLC) WITH
PTERIDINE
Fifty ~g of the purified 380:440/400:460 fluorophore/pteridine standards were dissolved in 0.5 mL of 0.1 M NaOH; 2 txL of the fluorophore//standards were applied to MN-300 and MN-300F cellulose plates and then developed in the following solvent systems (8,9): 3% (w/v) NH4C1; n-propanol:l% NH3 (2:1 v/v); butanol:acetic acid:water (4:1:1 v/v/v); isopropanol:2% NH 4 acetate (1:1 v/v); n-propanol:water (70:30 v/v); and ethanol:water (1:3 v/v). Plates were air dried, spots visualized by fluorescence detection and Rf values calculated. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
The purified fluorophore, pteridine standards, and patient samples were analyzed by reverse phase HPLC with fluorescence detection (Ex 370 nm/Em 418-700 nm) using IBM Cls (5 ~, 4.5 × 250 mm) column and a gradient system of 0.025 M triethylamine acetate buffer, pH 8.0 and acetonitrile (7). Chromatographic runs were performed in triplicate. Retention times of the fluorophore from purification/patient samples were compared to the standards. Quantitative data for the patient samples were obtained by comparison to a xanthopterin calibration curve. GAS-PHASE ACID HYDROLYSIS
The method was modified from Meltzer et al (10). Two mL of 6 N constant boiling HC1, 150 ~L of thioglycolic acid, and 20 ~L of liquified phenol were added to a 25 mL beaker and placed on the bottom of a glass desiccator. Twenty-five ~g of the purified fluorophore/pteridine standards and 20 ~L of HPLC grade water were added to a 3 mL amber vial. Two peptides, alpha MSH and oxytocin were treated in the same manner. Vials were placed in the desiccator using the ceramic plate as a holder. The desiccator was sealed, evacuated, and placed in an oven for 24 h at 115 °C. After acid hydrolysis, vials containing the peptides were analyzed for amino acid content (Model 119 CL Amino Acid Analyzer, Beckman Instruments, Inc., Palo Alto, CA, USA). Molar ratios were calculated to determine if acid hydrolysis was complete. The fluorophore/pteridine standards were analyzed by HPLC as described above. GAS CHROMATOGRAPHY-MASSSPECTROMETRY(GC-MS) The method is modified from that of Kuster and Niederwieser (11). A solution containing 100 ~L of
408
BSTFA and 100 ~L of HPLC grade acetonitrile was added to 50 ~g of purified fluorophore/pteridine standards in a Reacti-vial (Pierce Chemical Co., Rockford, IL, USA). The mixture was sonicated for 30 s, derivatized at 100 °C for 1 h, and then analyzed (within 2 h of silylation) by applying 2 pxL to a moving injector attached to a Finnigan Model 9610 gas chromatograph (Finnigan MAT, San Jose, CA, USA), fitted with a DB-1, (30 m, 0.25 mm i.d.) glass capillary column (J&W Scientific, Folsom, CA, USA). Analytical conditions were: column pressure -- 17.5 psi using helium (flow rate = 350 mm/s), and oven temperature program -- 180 °C isothermic for 3 min, then increments of 10 °C/min to 270 °C, followed by 270 °C isothermic for 3 min. Electron impact mass spectra were generated with a Finnigan Model 4500 mass spectrometer as follows: ionizing energy -- 70 eV, ion source temperature -- 170 °C, accelerating (electron multiplier) voltage -- 1.1 kV, scan range -m/z 50-700, and a scan time of 1.95 s/scan. DIRECT PROBE-MASS SPECTROMETRY
(DP-MS)
Mass spectra of the purified fluorophore/pteridine standards were determined by direct insertion probe using electron impact (EI) and chemical ionization (CI) by modifying the method of Blair and Foxall (12). Ten ~g of sample was dissolved in 10 ~L of methanol; 1 ~L of this solution was deposited onto a desorption wire and evaporated. Mass spectra (EUCI) were generated using a Finnigan Model 8430 Mass Spectrometer (Finnigan - MAT, San Jose, CA, USA) as follows: ionizing energy -70 eV, accelerating voltage -- 3 kV, ion source temperature -- 200 °C. CI analysis required 2% N H 3 in methane at source pressure of 1 torr. Peaks were analyzed with an accelerating voltage scan using perfluorokerosene as the internal reference standard. FOURIER TRANSFORM INFRARED
(FTIR)
SPECTROSCOPY
Infrared analysis was performed on crystals of the fluorophore/pteridine standards using an IBM Model 32 Spectrometer (IBM Instruments, Inc., Danbury, CT, USA), in conjunction with a Bruker Infrared microscope (Bruker Instruments, Inc., Billerica, MA, USA). Crystals of each sample were placed on an infrared window, examined for homogeneity by using the visible transmitting mode, and then directly bombarded with an infrared beam to generate the FTIR spectra. Results
Since the spectral characteristics of the 380:440/ 400:460 fluorophore were similar to the pteridines, thin layer chromatography was performed using pteridine standards on MN300 cellulose with six
CLINICAL BIOCHEMISTRY, VOLUME 24, OCTOBER 1991
XANTHOPTERIN IN CHRONIC RENAL FAILURE. II
RWValues
TABLE 1 ( × 100) of Pteridines and the 380:440/400:460 Fluorophore from U r e m i c H e m o f i l t r a t e
Using MN-300F Cellulose Thin Layer Chromatography Plates Solvents a
Compound b 6-Biopterin 6-Hydroxymethylpterin Isoxanthopterin Leucopterin Lumazine 6-Methylpterin L-Monapterin D( + )-Neopterin Pterin Pterin-6-Carboxylic Acid Xanthopterin Idione) Xanthopterin Monohydrate Pteroic Acid Folic Acid 380:440/400:460 Fluorophore
NH4C1
P-A
73 57 40 42 74 43 68 70 58 53 45 45 8 34 45
40 34 16 3 40 45 27 30 42 14 23 23 1 3 23
B-A-W 42 32 25 9 45 53 17 19 43 12 39 39 40 40 39
I-NHtAc
P-W
E-W
51 43 29 7 49 50 37 43 46 19 20 20 7 13 20
32 23 15 0 1 35 15 16 26 10 6 6 7 5 6
70 61 39 2 73 60 64 67 60 54 26 26 55 21 26
aNH4C1 -- 3% Cw/v) NH4C1 in distilled water; P-A = n-propanol-l% NHa ~2:1; w/v); B-A-W = butanol-acetic acid-water (4:1:1) by volume; I-NH4Ac = isopropanol-2% ammonium acetate Ii:l; v/v); P-W = n-propanol-water (70:30; v/v); E-W = ethanol-water (1:3; v/v). bConcentration = 100 p.g/mL. different solvents. The Rf values of the fluorophore and standards are given in Table 1; the R f v a l u e s of the fluorophore are identical with those of xanthopterin (dione/monohydrate) in all solvent systems. To confirm the similarity of the two compounds, analysis of the pteridine standards was performed using reverse phase HPLC with the triethylamine acetate/acetonitrile system. Only the retention time of xanthopterin was identical to t h a t of the fluorophore. Furthermore, no change in HPLC retention time was observed with either compound after gas-phase acid hydrolysis for 24, 48, and 96 h. These results indicate t h a t xanthopterin, like the 380:440/400:460 fluorophore, has unusual chemical stability in accordance with our previous findings (5). Since xanthopterin has a M r --- 179 Da, the Sephadex G-10 gel column was recalibrated using pteridine standards as molecular weight markers (Figure 1). It is clear from the chromatographic scan that: 1) xanthopterin has an elution volume very similar to the compound contributing to the 380:440/400:460 fluorescence, and 2) several pteridines do not elute in proportion to their relative molecular masses (Mr). Our previous results indicated t h a t the compound we isolated had a Mr 600 daltons (4). However, during our preliminary investigations, we were uncertain as to which class of compounds the fluorophore belonged. Consequently, pteridine standards were not used initially for calibration. This led to an erroneous estimate of the fluorophore's molecular weight.
CLINICAL BIOCHEMISTRY, VOLUME 24, OCTOBER 1991
The ultraviolet/fluorescent spectral characteristics of the fluorophore were also compared with t h a t of xanthopterin. Except for a slight difference in relative absorbance/emission intensity, the spectral curves were identical. Gas chromatography-mass spectrometry (GCMS) was used to confirm the purity of the fluorescent compound and compare its fragmentation pattern to t h a t of the xanthopterin standard. The purity, assessed by relative peak area, was shown to be 99% for the fluorophore. The EI mass spectra of the trimethylsilylated (TMS) derivatives of both compounds are essentially the same (Figure 2). The minor peaks at m/z of 73 (TMS-moiety), 147, and 171, as well as the major fragments at m/z 292, 308, 380 (base peak), and 395 (TMS-parent ion), are in accordance with those previously reported (13). The peak at m/z of 323 is due to a loss of one TMS group (73) from the parent ion (395). As with most TMS-pteridines, the base peak (380) is the M-15 fragment. The molecular weight of the compound (179) was determined by subtracting each trimethylsilylated moiety (73) from the parent ion (395), then adding a hydrogen (1) for every replaceable TMS-moiety (395 - 219 + 3 = 179). The EI mass spectra of underivatized fluorophore and xanthopterin using a direct desorption probe is depicted in Figure 3; again, both compounds have similar spectra. The characteristic fragments at m/z of 109, 151, and 152 are in accordance with those reported for underivatized xanthopterin and are due in part to the loss of hydrogen cyanide (HCN) and carbon monoxide
409
WILLIAMS, SHAYKH, AHMED, ETAL. I I I
o.16.
LEGEND: M O L E C U L A R W E I G H T ( M W ) M A R K E R S OB: D E X T R A N BLUE (MW 2x 106 ) PA: PTEROIC ACID ( M W 3 2 1 ) LUM: LUMAZINE (MW 164) CREAT: CREATININE (MW 113) NPT: NEOPTERIN (MW 2 5 3 ) BPT: BIOPTERIN (MW 2 3 4 ) XPT: X A N T H O P T E R I N (MW 179) 6 - C P : 6 - C A R B O X Y P T E R I N (MW 2 0 7 ) T R P : T R Y P T O P H A N (MW 2 0 4 )
i f il II
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EM 4 6 0 nm
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i 2's
~ so
;s
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l~s
. 120
ELUTION VOLUME
17s (mL)
2~0
2~5
2~0
2~5
Figure 1--Sephadex G-10 gel chromatogram of YC05 ultrafiltrate from uremic serum and hemofiltrate using ultraviolet detection at 280 nm (calibrated with pteridine standards). Shaded area indicates fraction containing 380:440/400: 460 fluorescence. (CO) from the parent molecule (12). The molecular ion (179) representing the molecular weight is also prominent. Confirmation of the molecular weight of both compounds was also accomplished by direct probe using chemical ionization (CI). Analysis by CI demonstrated identical fragmentation patterns generating the molecular weight plus one (M + 1) ion of 180. Thus, a molecular weight of 179 was confirmed for both compounds. Infrared analysis of the fluorophore and xanthopterin standard showed identical "fingerprint regions" (Figure 4). The following frequencies (wavenumbers) were shown to be characteristic of both molecules: 3300-3500 (amines, N-H stretch), 1690-1760 (ketones, C = O stretch), 1600 (aromatics, C = C), 1180-1360 (amines, C-N), and 675-870 (aromatic rings, C-H). Serum levels of xanthopterin from patients with chronic renal failure (CRF) and healthy subjects are given in Table 2. The levels observed in uremic patients are markedly elevated compared to individuals with no renal disease. These observations are in accordance with our earlier data pertaining to the 380:440/400:460 fluorescence. The large variation noted in the CRF population is expected with patients who are receiving maintenance hemodialysis.
Discussion The results of this study have shown that the elevated endogenous fluorescence at Ex 380:Em 440/Ex 400:Em 460 nm in patients with chronic renal failure is due to the unconjugated pteridine, 410
xanthopterin (2-amino-4,6-pteridinedione). Other compounds of high excitation/emission maxima were neither detected in other fluorescent/UV fractions nor in the pre/post column effluent during each analyical step. These results suggest that xanthopterin is the main contributor to the fluorescence observed at these high wavelengths. Although our initial studies, using acid hydrolysates of crude ultra filtrates, indicated that the fluorescence was not an intrinsic property of any peptide (5), we did report that the fluorophore appeared to be linked to a peptide containing: glycine, serine, aspartic acid, and glutamic acid (4). These amino acids persisted throughout the first two phases of HPLC purification, i.e., reverse phase followed by anion exchange. Since a basic solvent system (triethylamine acetate, pH - 8.0) was employed during our initial HPLC separation, a peptide containing the acidic amino acids, aspartate and glutamate, would most likely be negatively charged; so would the fluorophore (xanthopterin), with a pI - 4.0. Triethylamine, in this solvent system, is cationic and, thus, acts as an ion-pairing reagent for anionic compounds. Therefore, the separation on a reverse phase column would be based primarily on hydrophobicity. The purified fluorophore (xanthopterin) and this peptide appear to have a similar hydrophobicity index causing co-elution during analysis by reverse phase HPLC. Separation on an anionic column using a pH gradient would also most likely result in co-elution, since both compounds appear to have functional groups with pKas around 4 to 5. Acid hydrolysis of either HPLC fraction (from reverse CLINICAL BIOCHEMISTRY,VOLUME 24, OCTOBER 1991
XANTHOPTERIN IN CHRONIC RENAL FAILURE. II 5264
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500
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Figure 2-Comparison of electron impact (EI) mass spectrum of the 380:440/400:460 fluorophore and xanthopterin from gas chromatography using trimethylsilylated (TMS) derivatives. Base peak = fragment with greatest intensity; M + . = TMS-molecular ion. Arabic numbers represent the mass/charge ratio (m/z).
phase or ion exchange) would give similar results, i.e., a stable fluorescent product contaminated with amino acids. This effect would also be observed if the fluorophore was linked to a peptide containing these amino acids. Thus, the fluorophore, during the first two stages of HPLC purification, appeared to be linked to a peptide. If this were true, the retention time of the fluorophore from reverse phase HPLC would have most likely changed after acid hydrolysis. However, this was not the case, indicating that the fluorophore was not linked to any peptide. CLINICAL BIOCHEMISTRY, VOLUME 24, OCTOBER 1991
During our preliminary studies, it was also suggested that the fluorophore had a molecular weight of approximately 600 daltons (4), representative of the so-called "middle molecule" region (14,15). However, after identification, the fluorophore (xanthopterin) was shown to have a Mr = 179 daltons. Although these results appear discrepant, they are not unusual, considering the problems associated with gel chromatography. Several groups have demonstrated that many middle molecular substances isolated by this technique are really much smaller in molecular size (16-18). The discrepan411
WILLIAMS,
100-
SHAYKH,
AHMED,
ET
AL.
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Figure 3--Mass spectra comparison of the 380:440/400:460 fluorophore and xanthopterin standard using direct probeelectron impact (EI). M+. = molecular ion. Arabic numbers represent the mass/charge ratio (m/z). cies observed with this technique are due to the anomalous retention behavior of certain solutes isolated by gel filtration chromatography (17-19}. Aromatic and heterocyclic compounds tend to adsorb on the gel surface, giving elution volumes larger than expected from size exclusion curves. Ionic compounds, however, are often excluded from the gel particles due to charge interactions or the formation of solvation layers. Therefore, they often elute earlier than estimated. Xanthopterin, although a heterocyclic compound, is ionized at an alkaline pH. Since the pH of our buffer system during gel chromatography was 8.0, xanthopterin
would tend to elute earlier than expected. Another problem associated with gel filtration is its low resolving power, especially for low to middle molecular weight compounds. As shown in this study, this problem is also apparent with compounds such as xanthopterin and other pteridines. Although it is paramount to use calibrators representative of the class of compounds being analyzed, this may not initially be possible. Such was the case during our preliminary investigations of xanthopterin. Unlike the conjugated pteridines (folates), the function of most unconjugated pteridines (pterins),
XANTHOPTERIN IN CHRONIC RENAL FAILURE. II 1
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Figure 4--Comparison of the infrared spectra of the 380:440/400:460 fluorophore with a xanthopterin standard using fourier transform infrared (FTIR) analysis. including xanthopterin, has yet to be elucidated. So far, only biopterin has been shown to have a defined role as a cofactor in the hydroxylation of sevTABLE 2 Comparison of Serum Xanthopterin Levels (nmol/L) in Normal Subjects and Patients with Chronic Renal Failure
Group
Normal
CRF
n Range Mean SD
34 6-28 13 -+5
79 84-276 155 -+60
p < 0.001. CLINICAL BIOCHEMISTRY, VOLGME 24, OCTOBER 1991
eral aromatic amino acids which lead to the formation of neuronal hormones such as the catecholamines and serotonin. Several studies have shown altered catecholamine metabolism in uremia, suggesting that the unconjugated pteridines may play a role in the causation of some neurological symptoms (20,21}. Dhondt and Vanhille have reported an increase in serum pteridines from maintenance dialysis patients (22). Using HPLC with fluorescence detection, they demonstrated increased biopterin and neopterin levels compared to normal subjects. The increase paralleled the severity of renal failure. Other pteridines, however, were not examined. Our results indicate that xanthopterin is also significantly elevated. The increase in serum xanthopterin (as well as biopterin and neopterin) lev41.':1
WILLIAMS, SHAYKH, AHMED, ETAL. els m a y be directly r e l a t e d to r e n a l failure since the k i d n e y has an i m p o r t a n t role in r e g u l a t i n g p t e r i d i n e c o n c e n t r a t i o n s (23,24). H o w e v e r , xant h o p t e r i n is one of the end products of b i o p t e r i n and n e o p t e r i n m e t a b o l i s m (25). Thus, a n increase in c a t a b o l i s m of those compounds could also be a source of increased x a n t h o p t e r i n production. X a n t h o p t e r i n s t i m u l a t e s r e n a l cell mitosis in u r e m i c a n i m a l s (26); a f t e r a d m i n i s t r a t i o n , it induces h y p e r p l a s i a and h y p e r t r o p h y of the k i d n e y (26). Since the residual n e p h r o n s in m a n also hyp e r t r o p h y as r e n a l disease progresses, e l e v a t i o n s of x a n t h o p t e r i n m a y play a role in this m e c h a n i s m . Regardless of its etiology, we h a v e shown t h a t x a n t h o p t e r i n is m a r k e d l y e l e v a t e d in the sera of p a t i e n t s with chronic r e n a l failure. T h e p t e r i d i n e m a y be a b e t t e r m a r k e r of chronic r e n a l failure because of its h i g h stability and u n i q u e spectral properties (27). F r o m o t h e r studies, it a p p e a r s to play a major role in k i d n e y metabolism. H o w e v e r , additional work is n e c e s s a r y to e v a l u a t e its clinical significance and its role in the p a t h o g e n e s i s of the u r e m i c syndrome.
7.
8. 9.
10. 11. 12. 13.
Acknowledgements The authors wish to thank Dr. Jerry Hribar of the G.D. Searle Company and Dr. John Fitzloff of the University of Illinois for their technical assistance and advice regarding the mass spectrometry and infrared analysis studies. We would like to recognize the Graduate Resources Center at the University of Illinois for providing us with the necessary equipment to perform the gas chromatography-mass spectrometry analyses. We also wish to acknowledge Ann Poulos for excellent technical assistance with the amino acid evaluations. This study has been presented by Robert H. Williams as part of a thesis for the degree of Doctor of Philosophy at Rush University in Chicago and was supported in part by the Gwen Dubin Fund. We record with sadness the death of Professor Alvin Dubin before this paper could appear in print. The surviving authors wish to express their indebtedness to him as a teacher, a colleague and a generous personal friend.
14.
15. 16. 17. 18. 19. 20.
References 1. Swan JS, Kragten EY, Veening H. Liquid chromataographic study of fluorescent materials in uremic fluids. Clin Chem 1983; 29: 1082-4. 2. Barnett AL, Veening H. Liquid-chromatographic study of fluorescent compounds in hemodialysate solutions. Clin Chem 1985; 31: 127-30. 3. Shaykh M, Bazilinski N, McCaul DS, et al. Fluorescent substances in uremic and normal serum. Clin Chem 1985; 31: 1988-92. 4. Williams RH, Shaykh M, Poulos A, et al. Separation and purification of a fluorescent peptide from patients with chronic renal failure. Clin Chem 1988; 34:1276 (Abst.). 5. Williams RH, Dubin A, Shaykh M, et al. Biochemical elucidation and HPLC fractionation of fluorescent peptides in patients with chronic renal failure. Adv Exp Med Biol 1987; 223: 205-13. 6. Brunner H, Mann H. What remains of the "middle
414
21.
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