Pterins in human urine.

Abstract Qualitative and quantitative analyses for pterins (derivatives of 2-amino-4-hydroxypteridine) in human urine were carried out by an improved procedure which involved the use of two new weak ion exchangers, ECTEOLA-Sephadex and phospho-Sephadex. The predominant pterins found in urine were biopterin [6(l-erythro-dihydroxypropyl)pterin] and d-erythroneopterin [6(d-erythrotrihydroxypropyl)pterin], and daily individual outputs of these pterins were 980 µg and 380 µg, respectively. Crithidia fasciculata assays of urine revealed that 1.5 mg of biopterin equivalents were excreted daily. Oral administration of a large amount of folic acid did not affect the amount of these pterins. Furthermore, evidence was obtained which indicated that these pterins occur as the dihydro form. Additional pterins found in urine are l-threoneopterin [6(l-threotrihydroxypropyl)pterin], sepiapterin (6-lactyl-7,8-dihydropterin), pterin, and isoxanthopterin (7-hydroxypterin).

in human urine were carried out by an improved procedure which involved the use of two new weak ion exchangers, ECTEOLA-Sephadex and phospho-Sephadex.
Crithidia fasciculata assays of urine revealed that 1.5 mg of biopterin equivalents were excreted daily.
Oral administration of a large amount of folic acid did not affect the amount of these pterins.
Furthermore, evidence was obtained which indicated that these pterins occur as the dihydro form.
The published data on pterins in human urine (l-4) have been qualitative, so quantitative analysis of urinary pterins may give some information on the origin of pterins and on additional possible metabolic roles of pteridines in man.
In this paper, an improved method of quantitation of urinary pterins is described.
By the use of the method, the effect of dietary high folate on urinary pterins was examined.
Pterins which had not been detected previously in human urine have been isolated and identified.
Applicability of the method and the possible origin of pterins in human urine are discussed.
Isosepiapterin was made from dihydrobiopterin by a modified method of Katoh and Akino (12). One micromole of dihydrobiopterin was dissolved in 5 ml of distilled water and the solution was applied to a phospho-Sephadex column (Hf, 1.5 x 5 cm). After the development with a few milliliters of distilled water, the column was allowed to stand overnight in the dark at room temperature.
The development of the column with water was resumed the next day, and two fluorescent peak fractions, yellow and blue, were obtained.
The yellow fluorescent compound moved as a single band or spot when subjected to column or paper chromatography.
RF values of the compound were similar to those of isosepiapterin in five solvent systems. The yield of isosepiapterin was about 20%. Almost all of the remaining dihydrobiopterin was recovered as biopterin. Pterin, 6-hydroxymethylpterin, B-carboxypterin, xanthopterin, and isoxanthopterin were obtained from commercial sources. Lumazine was kindly supplied by Dr. C. 11. Baugh. Sephadex G-25 and G-10 were obtained from Pharmacia.
ECTEOLAcellulose was purchased from Sigma and prepared for use by treating with NaCl-HCl solutions (9). Other chemicals were obtained from commercial sources.
These Sephadexes can be used repeatedly after mashing with some loss in capacity.
Since the OH form of ECTEOL;\-Sephades is too strong for the purificntion of yteridines, the absorptivity was reduced with HCl as was done on ECTEOLA-cellulose (9). Moistened ECTEOLS-Sephades (OH) was suspended in 5 volumes of 5 msr YaCl solution. The pH of the suspensiotl ~vas approximately 10.5. To the suspension, 1 K HCl was added dropwise with continuous stirring and the addition of HCl was stopped at p1-I 8, 7, or 6, and the Sephadex was washed with water on a glass filter. ECTEOLA-Sephadeses thus treated are called "pH 8, 7, or 6", respectively. P-Sephadex in the Hf form \Tas prepared for chromatography by washing with 0.1 s HCl and water. Generally, columns of these Sephadexes were developed with mater. Chromatographic data of several pteridines and purines are shown in Table I. Most of these pteridines can be separated from one another by use of these two column systems. Results are similar to those obtained with corresponding cellulose exchangers (14, 15). The merits of these Sephadexes over the corresponding cellulose types are translucency and homogeneity. Another advantage is that P-Sephades is superior to phospho-T~BLIZ I Column chromatography of pteridines and pwines on ECTEOLA-Sephaclex and P-Sephaclex columns A mixture of two to five compounds was applied to a column (1.1 X 20 cm) and developed with water.
The fractions (2 or 5 ml) were collected at a rate of 0.5 ml per min and analyzed by ultraviolet absorption or fluorescence. cellulose in the separation of biol)terin from n-erythroneopterin. The relative elution volume of u~eryfhrolieopterill in the Sephades type was 93 (Table I), whereas that in the cellulose type was 98 (15).

Paper
Chro,,zafoqraphll-Palled clll,on,atograpliy was carried out in the dark at' room temperature by the ascending procedure. Rhatman No. 20 paper was used because it gives generally good separation of pteridines.
The bioassay was performed according to the procedure of Dewey and Kidder (16). The results of unknown pterins were compared with those of authentic pterins.
Quanfitafive _If easurement of Pteridines-The amounts of pterins were determined by use of the extinction coefficient for each pterin (9,17,18 ter equipped with a primary filter exciting at 360 ml ad a secordary filter emitting wave lengths greater than 415 inn was used. The fluorescence properties are shown in Table III. IIuman Urine-Urine samples for quantitative analysis were kindly supplied by Dr. Robert 1I. Herman of the United States Amy Uedical Research and Nutritional Laboratory. "Normal urille" 1vas rollect,ed from four individuals who llatl been receiving 100 pg of folic acid with a defined diet daily.
"IIigh folate uriue" wax collected from the same indi\-iduala after consuming 15 mg of folic aci(l per day for 3 dxyY. Kriue for qualitative analysis was collected at this laboratory.
Freshly voided urine TWS froze11 and stored at -20" until use.

RESULTS
hll experiments were carried out in a darkened u)om, and saril)les aud fractions were stored at -20" bet\veen purification steps and analyses, or both.

Quantitative Ilnal&s 01 Pterins in Urine jrom Subjects Receiving a Arormal Level of Folate
The 24.hour output from four individuals receiving 100 pg of folic acid per day was pooled, :nld 1 y0 (57 ml) was prepared for quantitative yterin analyses.
The mixture was acidified with the addition of 2.5 ml of 6 x HCl (pfl N l), and the solution was applied to a Dowex 50 colurm (Hf, 2.5 x 6 cm) (19). After the column was washed with about 60 ml of water, the pteridines \vhich rvere retained on the resin \vere recovered by elution with about' 60 ml of 1 N NIl,OII.
The eluate was concentrated to about 5 ml by a rotary evaporator and the concentrated solution was applied to a pH 7 ECTEOL.~-Sephadex colu~ml (2.5 x 11 cm).
TWO fluorescent peaks lvere obtained by tleveloping the column with water.
The column ~1s further developed, first with 0.5 s acetic acid and then lvith 0.1 s 11Cl. The elution profile is presented in Fig. 1. Peak 1 showed yello&h-blue column. The column size was 2.5 X 11 cm. The column was developed with water, 0.5 N acetic acid, and 0.1 N HCl successively, and 15-ml fractions were collected.
Fluorescence was measured after dilution (160fold). The amount was expressed as arbitrary units. fluorescence; Peak II, blue; Peak III, purple; Peak IV, whitishblue. Fractions of each peak were combined, concentrated, and subjected to further purification.
Pea72 I: Sepiapterin, Isosepiapterin, A'anthopterin, and Biopterin-Materials from Peak I \Tere applied to a Mephadex column (1.1 x 19 cm), and the column was developed with water. TWO yelloIv fluorescent peaks and one blue fluorescent peak Tvere obtained.
The first yellow fluorescent fraction contained riboflavin as determined by P-Sephadex and paper chromatography.
Since a clear separation of the second peak (yellow fluorescent) and the third peak (blue fluorescent) was not obtained, all of the fractions containing these two peaks were combined and rechromatographed on a longer PSephadex column (0.9 x 40 cm). A bluisll-green fluorescent substance was eluted between 20 and 25 ml of water; yellow Huorescent substances between 35 and 55 ml; blue to green fluorescent substances bet\veell 55 and 75 ml; blue fluorescent substance between 110 and 140 ml. Each fraction was concentrated and analyzed by paper chromatography. The fluorescent substance iu the first peak fraction (20 to 25 ml) did not appear to be a pteridine because of its high RF ralues in several solvent systems. Paper chromatography ii1 fi\-e solvent systems showed that the second fraction (35 to 55 ml) contained sepiapterin, isosepiapterin, and other yellow lluorescent substances, and the third fraction (55 to 75 ml) colltsLined xanthopterin.
The blue fluorescent substance in the fourth fraction (110 to 140 ml) was pure as determined by paper chromatography and the mobility was the same as that of biopterin.
The amount was estimated to be 5.5 pg.
Peak II: Pterin, Erythroneopterin, Biopterin, and Threoneopterin-Material from Peak II was applied to a pT-I 7 lKX'EOLh-Sephndex column (0.9 x 41 cm), and, by development with water, the blue fluorescent peak Jl-as resolved into two zones. A major fluorescent fraction K:M eluted between 105 and 135 ml and a minor fluorescent fraction was obtained bet\veell 140 and 165 ml. Each peak fraction n-as concentrated.
The minor blue Auorescellt fraction was apl)lietl to a 1'.Sephadex column (1.1 x 19 cm) and was eluted as a single peak from the column.
Behayior of this substance 011 EC'I'EOL~~-Sephades, Mephadex, and paper (five solvent systems) was similar to that for pterin. The amount of the substance was 2.9 pg.
The major fluorescent substance was then applied to a long P-Sephadex column (1.2 x 110 cm). The column was developed with water and IO-ml fractions were collected.
As shown by the solid circles in Fig. 2, the major fluorescent substance was resolved into two peaks. The elution volumes of the first peak and the second peak are the same as that obtained with known erythroneopterin and biopterin, respectively. These results mere confirmed by paper chromatography of each peak in five solvent systems. The amount of erythroneopterin found Teas 15.4 pg and biopterin 33.5 pg. In the second peak which contained biopterin, another minor compound was detected.
The compound was separated from biopterin by paper chromatography (Solvent U). The isolated minor compound then migrated as a single spot, similar to a threoneopterin on paper in each of five solvent systems. The amount was approximately 1 pg.
The occurrence of biopterin in Peaks I and II was unexpected. Subsequent chromatography by P-Sephadex alters any dihydrobiopterin present to biopterin or isosepiapterin.
Additional observations regarding dihydropterins are presented in Experiment 5.
Peak 111: 1sozanthopterin-The pooled fractions of Peak III from the first ECTEOLA-Sephades column (acetic acid eluate) were dried by a rotary evaporator, and the residue was dissolved with a small amount of water.
The solution was fractionated on a P-Sephadex column (1.1 x 25 cm). A purple fluorescent fraction (20 to 50 ml) was eluted first followed by blue fluorescent fractions.
The purple fluorescent fraction was concentrated and applied to a pH 6 ECTEOLASephadex column (0.8 x 11 cm). The column was developed with water and yielded four fluorescent zones. The first zone, eluted between 40 and 50 ml, showed blue fluorescence, and the second, eluted between 60 and 75 ml, showed purple fluorescence.
The second fraction was identified as isoxanthopterin (paper chromatography). The amount was 0.9 lg. The substances in the first zone and in the other two zones still remaining in the upper part of the column were not identified.
Peak IV: 6-Carbozypterin-Peak IV, the HCl eluate, was dried, and the residue was dissolved in water and applied to a P-Sephadex column (2.5 X 12 cm). ,411 of the blue fluorescent fractions which were eluted first were combined, concentrated, and streaked on Whatman No. 20 paper (35 x '35 cm). The paper was developed iu Solvent D. The area corresponding to 6-carboxypterin was cut out, eluted with water, and the eluate was rechromatographed in Solvent E. The fluorescent band was again eluted, and the eluate from the paper was passed through a short P-Sephadex column (0.8 X 5 cm). The 6carboxypterin thus purified was estimated to be 0.5 pg. The results are shown in Table IV isoxanthopterin, and pterin were also detected, but the amount of these pterins was small. Crithidia activit) of whole urine revealed that about 60 pg of biopterin equivalents were present in 57 ml of the urine mixture.

Experiment 2 Recovery Test
For the evaluation of the recovery of pterins from urine and the reproducibility of the data in Experiment 1, 24.5 fig of authentic n-erythroneopterin were added to 57 ml of normal urine and the mixture was analyzed as in Experiment 1. The results from the step using the long P-Sephadex column chromatography which separates erythroneopterin from biopterin are presented in Fig. 2. The increased amount of Peak I material found in Experiment 2 versus Experiment 1 is reflective of the added erythroneopterin to the urine sample, whereas the biopterin peak remained unchanged.
The quantitation of these pterins and others are summarized in Table IV, Experiment 2. The recovery of the added erythroneopterin was calculated to be 82%, and the amounts found for other pterins was in good agreement to those found in Experiment 1.

Experiment 3 Eflect of High Folate Diet on Urinary Pterins
The effect of high folate intake on urinary excretion of pterins was studied.
9 urine mixture of 1 v. each of the 24.hour output from four individuals receiving 15 mg of folic acid was prepared. The volume of the pooled urine was 77 ml, and the urine sample was analyzed by the same procedure used in Experiments 1 and 2. Results given in Table IV processed by a procedure similar to Experiment 1 to obtain about 10 pg of threoneopterin.
Each of these pterins was subjected to ECTEOLA-cellulose sodium borate column chromatography, and fractions were assayed for Crithidia activity. Fig. 3A illustrates the chromatographic elution profile of authentic pterins and Fig. 3B is the elution profile of purified urinary pterins as biopterin, erythroneopterin, and threoneopterin. (2.5 x 3 cm). After the column was washed with water, the pterin was recovered by 1 N NI&OH. The eluate was concentrated and purified by a P-Sephades column (0.8 x 5 cm). Crithidia growth factor activity of threoneopterin purified in this way was examined by the system of Dewey and Kidder (16). Fig. 4 shows that the threoneopterin from urine supported the growth of Crithidia similarly to L-threoneopterin.
Hence, these results suggest that the configuration of the side chain is L-three. Crithidia activities of urinary biopterin and erythroneopterin were also assayed and found to be similar to those of authentic biopterin and n-erythroneopterin, respectively. These are consistent with the data of Patterson et al.

Experiment 5 Dihydropterins in Urine
In Experiment 1, biopterin was isolated from Peaks I and II of the pH 7 ECTEOLA-Sephades column chromatography step. According to the elution volumes for the pteridines presented in Table I, dihydrobiopterin should be contained only in Peak I and biopterin in Peak II. Since dihydrobiopterin is converted to isosepiapterin and biopterin on a P-Sephadex column as is shown under "Materials and Methods," the presence of biopterin and isosepiapterin in Peak I suggests the occurrence of dihydrobiopterin in the urine. Accordingly, an experiment was performed to isolate and identify dihgdropterins in urine. In order to isolate and identify dihydropterins from urine, the procedure was modified by using Sephadex column chromatography which had been shown to be effective (9,17,22,23) instead of Dowex 50 and P-Sephadex chromatography.
To minimize spontaneous oxidation of dihydropterins, prolonged storage of samples was avoided even at -20".
The frozen urine was thawed and the precipitate was removed by centrifugation at 5". The supernatant fluid (200 ml) was applied to a Sephadex G-25 column (fine grade, 7 x 25 cm). The column was developed with water, and 20.ml fractions were collected. Salts (;ZgNOa precipitable substances) were eluted in fractions 41 to 54. A dark brown material lvas eluted in fractions 6'7 to 70. Just after these brown r FRACTION NUMBER FIG. 5. Elution profile of dihydropterins of the urine from pH 8 ECTEOLA-Sephadex column. The column (2.5 X 28 cm) was developed with water and 5-ml fractions were collected. Absorption at 330 nm corresponds to violet fluorescence under ultraviolet light at 365 nm.
fractions, most of dihydrobiopterin emerges, and fractions 71 to 84 were collected, combined, and concentrated.
During concentration by rotary evaporation, care was taken to avoid drying the sample. A total of 1 liter of urine was subjected to the chromatography.
The combined concentrate (50 ml) was applied to a Sephadex G-10 column (5 x 22 cm). The column was developed with water, and 13-ml fractions were collected.
Fractions 40 to 60 were combined and concentrated. This combined fraction should contain any dihydrobiopterin present judging from a chromatography of authentic dihydrobiopterin. The concentrated solution from the G-10 column was applied to a pH 8 ECTEOLA-Sephadex column (2.5 x 28 cm). The column was developed with water, and 5ml fractions were collected.
The elution pattern is shown in Fig. 5. Although a violet fluorescent substance was eluted as a single peak, some other ultraviolet light absorbing peaks were observed. Dihydropterins in the violet fluorescent peak were estimated to be about 0.5 mg from the absorption at 330 nm. This peak appeared to contain two violet fluorescent compounds.
Fractions 33 to 42 were combined, concentrated, and streaked on paper (35 x 35 cm). By developing the paper in Solvent B under an atmosphere of argon overnight, three fluorescent bands, one major (I) and two minor ones (II and III), were obtained.
Band I (RF 0.14), Band II (RF 0.07), and Band III (RF 0.28) corresponded to the mobility of dihydrobiopterin, erythrodihydroneopterin, and biopterin, respectively (Table II). Each fluorescent band was eluted with water by a descending procedure under argon gas.
The eluate from Band I was then applied to a pH 8 ECTEOLA-Sephadex column (0.8 x 8 cm), and a violet fluorescent fraction was collected.
This procedure clearly separates dihydropteridines from the oxidized forms. The ultraviolet absorption spectra of the violet fluorescent fraction were identical with those of 7,8-dihydrobiopterin which showed the characteristic bathochromic shift of 7,8-dihydropteridines ( Fig. 6) (24). As is shown in Fig. 7 7. Paper chromatograms of dihydrobiopterin from urine. A to E are the solvent systems described in the text. 1, dihydrobiopterin from urine; 2, authentic 7,%dihydrobiopterin.
Dihydrobiopterin from urine was spotted on paper just after the elution from the pH 8 ECTEOLA-Sephadex column. Crystalline dihydrobiopterin was dissolved just before it was applied to the paper. After development', dihydrobiopterin on paper was oxidized in the air. The photograph was taken under ultraviolet light (365 nm) by use of a filter (460 nm). The picture is a negative and the fluorescent spots are shown as black spots. results indicate that the violet fluorescent compound from Band I is 7,8-dihydrobiopterin.
The eluate from Band II was treated similarly to Band I. The ultraviolet absorption spectra of the violet fluorescent fraction from Band II were essentially the same as those shown in Fig. 6 This indicates some oxidation of dil~ydrobiopteri~~ to biopterin during concentration and paper c~llromatograph~.
Since selliapterin and isosepiapterin should be eluted a little later than tiih~-drobiol)terin and dihydroneopterin from the Sephades columns (15), fractions 84 to 100 from the Sephadex G-25 column and fractions 61 to 80 from the Sephadex G-10 column were combined and concentrated.
The concentrated solution was applied to a pH 7 ECTEOLA-Sephades column (2.5 x 10 cm.). On elution with water, a yellow Fluorescent substance was obtained in fractions where sepiapterin and isosepiapterin were expected to appear.
The yellow sub&lice was further purified by a P-Sephadex column, and the eluate was concentrated and applied to paper (35 x 35 cm). .$fter development in Solvent B, two yellow fluorescent bands were obtained.
The RF of the upper band was similar to that of sepiapterin and the RF of the lower band was about half that of the upper one.
The yellow fluorescent compound of the upper band was eluted with 0.1 N acetic acid, and the acid in the eluate was removed by passing it through a pH 7 ECTEOLA-Sephadex column (2.5 x 5 cm). The yellow fluorescent compound in the final eluate was pure as judged by paper chromatography; it migrated as sepiapterin in each of the five solvent systems. The ultraviolet absorption spectrum in water was also similar to that of sepiapterin. Therefore, the yellow fluorescent compound was concluded to be sepia.pterin.
The amount was 3 pg. The yellow fluorescent compound in the lower band appeared to be somewhat similar to a, yellow compound which was formed from dihydroneopterin (al), but further analysis was not made. There was no evidence for the occurrence of isosepiapterin.
The finding of isosepiapterin in Esl)eriment 1, therefore, n-as concluded to be an artifact due to dehydration of dihydrobiopterin.
From the data in Table IV,  .1t the 111-I 8 ECTEOLh-Sephades step in Experiment 5 ( Fig.  6), about half of the total pterins in urine (0.5 mg per liter) were detected as dihydropterins.
In consideration of t,he instability of dihydropterins, a much larger portion may be present as reduced derivatives.
The occurrence of reduced pterins has been reported in several organisms; dihydrobiopterin in rat (5) and Ascaris (23), tetrahydrobiopterin derivatives in ;1nacystis ni dulans (25), insects and Amphibia (26), and sepiapterill in insects (27)) fishes and Amphibia (28). The coenzyme forms of pterins, both naturally occurring and synthetic, have been demonstrated to participate in hydroxylation reactions as their reduced forms (5,8). Furthermore, most of their reactions require them to be reduced (18,29). Hence, it can generally be assumed that pterins are metabolically active in a reduced state.
Since a large portion of pterins is present as labile reduced forms in urine, degradation or conversion of reduced pterins to other pterins m2ty ha\-e taken place during isolation.
As has bee11 mentioned in the prel-ious section, isosepiapterin in Esperi-ment 1 is an artifact.
6-Carboxypt,erin may also be an artifact. Blair (3) detected 6-carboxypterin in human urine only when urine was allowed to stand or the preliminary stages of the analysis were protracted.
Reduced pterins were converted to santhopterin noneilz~matically as well as enzymatically (30). Therefore, it is not certain whether santhopterin is present in urine or not. As isoxanthopterin is formed from pterin enzymatically (31) both pterins seem to be present in urine.
It has been reported that sepiapterin reductase was detected in several animals (32, 33). Hence, it is probable that a small amount of its substrate, sepiapterin, is excreted in urine.
Although biopterin is probably an essential substance for the biosynthesis of adrenalin and serotonin, its origin in higher animals is not clear. Biopterin may arise in three ways in higher animals.
(a) It is taken in as a Titamin; (b) it is synthesized from other pterins such as folate; (c) it is synthesized from a nonpteridine precursor.
Our results on the urinary excretion of pterins indicate that high folic acid intake does not affect the level of biopterin and neopterins.
According to Goodfriend and Kaufman (34), a folate deficient diet failed to reduce the hepatic level of cofactor for phenylalanine hydroxylase (presumably reduced biopterin) in rat except when inanition was also induced. Also, in the case of germ-free chicks, the amount of ingested folic acid did not have an affect on the Crithidia activity of serum, and in the case of man, Crithidia activity and the folate level in serum were not correlated with each other (35). These results may exclude a precursor role of folate for biopterin in these animals. Pabst and Rembold (36) proposed a de nova synthesis of biopterin in the rat based on the observations that biopterin output was not altered by the administration of antibiotics or by changing the amount of dietary folic acid or riboflavin, and that biopterin n-as excreted constantly by rats grown on a biopterin-free diet for two generations.
As yet, definitive data about biopterin synthesis in man are lacking.
-%s we have shown, pterins with three carbon side chains in human urine are biopterin, n-erythroiieopterin, L-threoneopterin, and sepiapterill.
Recently, Fukushima (15) proposed that the intermediary metabolites of the biosynthetic pathway from GTP to biopterin are n-erythrodihydroneopterin, L-threodihydroneopterin, and sepiapterin.
These three pterins which are the constituent pterins in urine may suggest the involvement of these metabolites in the biopterin biosynthetic pathway.