Formation and Excretion of Pyrrole-Zcarboxylic Acid WHOLE ANIMAL AND ENZYME STUDIES IN THE RAT*

SUMMARY A corrected method for the measurement of pyrrole-Z-carboxylate in rat urine was used in studies of its excretion under various experimental conditions. The findings impli-cated administered hydroxy-L-proline as a relatively efficient source of urinary pyrrole-2-carboxylate and tended to exclude administered L-proline as a significant direct source. Removal of aerobic gut flora had no influence on the excretion of pyr-role-2-carboxylate either endogenously or following hydroxy-L-proline administration. Related studies showed that rat kidney L-amino acid oxidase catalyzes oxidation of hydroxy-L-proline to Al-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole3-carboxylate on acidification of reaction mixtures. All findings were consistent with hydroxy-L-proline as the source of endogenous pyrrole-2-carboxylate excretion. Excretion patterns and labeling patterns were compared after administration of pyrrole-2-carboxylate or of hydroxyproline epimers. From these data, the true excretion product of hydroxy-L-proline oxidation by L-amino acid oxidase appeared to be the unstable oxidation product, A’-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-car-boxylate in urine.

Pyrrole-2-carboxylic acid, as a compound of biological interest, was isolated after chemical degradation of sialic acids (3) and was subsequently identified as an acid-catalyzed dehydration product of Ai-pyrroline-4-hydroxy-2-carboxylate (4). The latter compound is formed by the oxidation of n isomers of hydroxyproline, catalyzed by mammalian n-amino acid oxidase (4) or by a more specific allohydroxy-n-proline oxidase of Pseudomonas (5). That pyrrole carboxylate can be derived from Damino acid oxidase action on hydroxyproline provided an explanation for the appearance of pyrrole carboxylate in rat or human urine after administration of the D isomers of hydroxyproline (6).
From a later observation in rats (7)) that radioactive L-proline also labeled urinary pyrrole carboxylate, the pathway, L-proline + hydroxy-L-proline --) pyrrole carboxylate, was inferred (7). However, no metabolic reaction was then known in animal tissues to account for the conversion of hydroxy-n-proline to the cyclic ketimine, Ai-pyrroline-4-hydroxy-2-carboxylate.
In animal tissues, rather, the principal fate of hydroxy-L-proline has been defined as oxidation to the cyclic aldimine, Ai-pyrroline-a-hydroxy-5-carboxylate (8), and subsequent reactions of this intermediate (9,10). Hypothetical explanations for pyrrole carboxylate formation from L-proline also included the possibility that L-proline itself might be the source, via a direct pathway independent of hydroxyproline, or that hydroxy-L-proline formed from L-proline could be converted to pyrrole carboxylate either by an undefined reaction in animal tissues, or by a route involving the intestinal flora (9).
In a recent brief publication (ll), we reported that in rats pyrrole carboxylate is labeled more directly from hydroxy-Lproline than from n-proline, and that a demonstrable enzymatic basis for this reaction was the oxidation of hydroxy-r-proline to the cyclic ketimine by mammalian L-amino acid oxidase of kidney; the latter finding was independently confirmed in a subsequent report (12). Our report (11) also presented data for the urinary excretion of pyrrole carboxylate by man; our values were in close agreement with less extensive studies published earlier (13,14).
Subsequent to that publication, we learned that our measurements of pyrrole carboxylate levels in both rat and human urine were in error because they included nonspecific compounds that had not been removed entirely either by our procedure, or by those procedures employed by other laboratories (13,14). An account of the methodologic problems and of corrected values for human urinary pyrrole carboxylate is published separately (15).
The present paper provides detailed data, using the corrected method, on the pattern of excretion of free pyrrole carboxylate under various experimental circumstances. While qualitatively the broad conclusions based on the less specific method (11) remained valid, the selective role of hydroxy-L-proline as a source of pyrrole carboxylate has emerged more clearly. In addition, kinetic data are provided for the oxidation of hydroxy-L-proline to the cyclic ketimine by tissue homogenates in several species and by the homogeneous L-amino acid oxidase of rat kidney.
Two new metabolic inferences concerning pyrrole carboxylate have emerged from the work reported here. One is the conclusion that urinary pyrrole carboxylate of endogenous origin, or that labeled from administered proline or hydroxyproline, is probably formed from the unstable pyrroline precursor in the urine rather than in the tissues of the animal. In contrast, administered pyrrole carboxylate is extensively conjugated and otherwise metabolically altered. Another inference is that allohydroxy-n-proline, (or its pyrroline oxidation product or both) is metabolized by the whole animal via an unknown route in addition to that forming pyrrole carboxylate.
Experiments designed to investigate the role of proline as a pyrrole carboxylate precursor have provided new information on the effect of a proline load on free hydroxyproline of rat liver and on the competition by proline for the oxidation of hydroxy-Lproline via the major pathway through Al-pyrroline-3-hydroxy-5-carboxylate. EXPERIMENTAL  ; L-proline and hydroxy-L-proline contained, respectively, less than 4 X lo-7 and 6 X 10-T molar equivalent of pyrrole carboxylate. L-Proline by asimilar technique (addition of radioactive hydroxy-L-proline and separation and measurement of the latter by ion exchange chromatography) contained less than 8 X 10-e molar equivalent of hydroxyproline.

Studies with Intact Rats
Excretion o j Pyrrole Carboxylate from Endogenous Sources-Table I shows values obtained by Method 2 for the daily excretion of pyrrole carboxylate in several groups of rats. The mean value, 0.012 pmo1/24 hours/100 g, is about 3-fold less than that earlier reported using a less specific method (11).
Effect of a Hydroxyproline or Proline Load-The data shown in Fig. 1, obtained by the revised pyrrole carboxylate method, indicate more than a 30-fold increase in pyrrole carboxylate excretion after large hydroxy-n-proline loads. Similarly administered Lproline produced at most a a-fold increase in pyrrole carboxylate excretion. It is of particular interest that, of the several pyrrolereactive components eluted from the amino acid analyzer column in the more specific method (15), only the true pyrrole carboxylate peak was increased by hydroxyproline or proline administration, implying that the other quasi-pyrrole carboxylate peaks were metabolically unrelated to pyrrole carboxylate. It therefore was apparent that at the higher levels of true pyrrole carboxylate measured in this kind of experiment, Method 1 and Method 2 would give very similar values.
Cowersion of Radioactive Hydroxy-L-proline and L-Proline to Pyrrole Carbosylate-Earlier data based on the less specific method for pyrrole carboxylate isolation indicated that administration to rats of either radioactive n-proline or hydroxy-n-proline resulted in excretion of labeled pyrrole carboxylate; radioactivity in pyrrole carboxylate represented an approximately IO-fold greater fraction of the dose of hydroxyproline than of proline. Repetition of these experiments with the more discriminating method revealed the presence of labeled impurities which separated from pyrrole carboxylate on the amino acid analyzer column (Fig. 2). Such labeled impurities were much greater after administering radioactive proline than after radioactive hydroxy- 1. Increased excretion of pyrrole carboxylate (WA) following hydroxy-L-proline or proline loads. L-Proline (0) or hydroxy-L-proline (0) was administered subcutaneously in four to five divided doses over 8 hours to groups of rats (4-G) for each dose level. Urine was collected for 24 hours and assayed for pyrrole carboxylate by Method 1 (high values) or by Method 2: as discussed in the text, at high values of pyrrole carboxylate Methods 1 and 2 give similar results. The ordinale shows -fold increase over the endogenous pyrrole carboxylate (PCA) excretion (0.012 ~01/24 hour/100 g, Table I) ; the abscissa shows proline or hydroxyproline adminis-&red per .iOO g body weight. A control experin%nt excluded the Dossibilitv that excreted hvdroxvnroline might vield significant IncreasesIn pyrrole carboxylate dy' conversion in"the urine. When urine from eight rats was collected for 24 hours in the presence of 2 mmol of hydroxy-L-proline (the expected excretion ratio for a load of 2.5 mmol per 100 g), the urinary pyrrole carboxylate was elevated only 30yo over the basal level.
L-proline (Fig. 2). The corrected recovery of radioactive pyrrole carboxylate after L-proline or hydroxy-L-proline administration is shown in Table II. The per cent of administered radioactivity recovered in true pyrrole carboxylate from hydroxy-L-proline was 80-to 200-fold greater than that from proline. The specific activity of pyrrole carboxylate in urine seemed significantly higher (3-to 4-fold) than that of free hydroxyproline in urine, whether proline or hydroxyproline was the radioactive compound administered (Table II). These values correct the much greater apparent discrepancy in specific activities, after administration of radioactive proline, reported on the basis of the less specific method for pyrrole carboxylate ( 1).
Pyrrole Carboxylate Excretion after Pyrrole Carboxylate Administration-Following intraperitoneal administration to rats of carboxyl-labeled [14C]pyrrole carboxylate (10 mg), Letellier and Bouthillier earlier reported that one-quarter to one-third of urine radioactivity was free pyrrole carboxylate, a large fraction being distributed between the glycine and glucuronic acid conjugates of pyrrole carboxylate (27). In our experiments, using Method 2, administration of 3 pmol of unlabeled pyrrole carboxylate to each of six rats resulted in the recovery of only 6.7 % of the dose as free pyrrole carboxylate with no accompanying increase in the pyrrole-reactive components which separate from true pyrrole carboxylate on the amino acid analyzer column (15).
[2-14C]Pyrrole carboxylate (2.2 x lo5 dpm per rat) was administered intraperitoneally in three separate trials involving groups of 6 to 14 animals. The fraction of the dose excreted in the first 24-hour period ranged between 71 and 85yo. Of the total radioactivity in urine, 86% (in each of the three trials) was retained in the aqueous phase after ether extraction. Essentially all of the radioactivity in the ether extract was recovered from the Dowex 50 column effluent (Method 1) and on further fractionation by the amino acid analyzer method (Method 2), was distributed between two peaks shown in Fig. 3A. The peak of free pyrrole 2. Elution pattern of urinary radioactivity and carrier pyrrole carboxylate after administration of radioactive L-proline or radioactive hydroxy-L-proline.
A, administration of 5.9 X 10' dpm of hydroxy-L-[G-3Hlproline to eight rats. The sample, isolated by Method 1, containing 2.3 X 106 dpm and 0.5 pmol of added carrier pyrrole carboxylate, was eluted from the DC-1 resin column as described earlier (15). B, administration of 5.5 X lo* dpm of L-[u-'%]proline to eight rats. The sample, isolated by Method 1, containing 6.2 X 10' dpm and 0. To investigate the possibility that the pyrroline product is consumed in reactions other than that producing pyrrole carboxylate, both radioactive and unlabeled allohydroxy+proline, in separate experiments, were injected subcutaneously in rats. After administering 20 pmol of unlabeled allohydroxy-n-proline to each of six rats, -urine was collected for 24 hours and the free pyrrole carboxylate and urinary hydroxyproline were measured. The total hydroxyproline excreted was 90 pmol, or 75% of the total amount administered. Free pyrrole carboxylate accounted for only 4.5 pmol, so that the 25 pmol unaccounted for could have been conjugated or metabolically altered pyrrole carboxylate.
To investigate this possibility, eight rats were injected subcutaneously with a total of 2.7 x 108 dpm of allohydroxy-n-[G-aH]proline. Urine collected during the next 24 hours contained 51 y. of the administered radioactivity. Of total urine radioactivity, the ether extract contained lo'%, all of which appeared to be in the pyrrole carboxylate peak (Fig. 3B). Examination of the aqueous phase following ether extraction indicated that essentially all the radioactivity was in residual allohydroxy-n-proline; hydroxy-L-proline accounted for less than 1 y. of the aqueous radioactivity. A small fraction of radioactivity in the aqueous phase 0 Number of rats in each experiment is shown in parentheses. b In control experiments, radioactive hydroxy-L-proline was added to rat urine and immediately processed as for pyrrole carboxylate isolation (Method 2), or was allowed to incubate in rat urine for 24 hours before determination of radioactivity in pyrrole (5% of that in the urine) could have been unextracted pyrrole carboxylate or tritiated water,' but there was no significant radioactivity attributable to water-soluble pyrrole carboxylate conjugates. In contrast, after administration of radioactive pyrrole carboxylate, most of the urine radioactivity was not etherextractable (presumably conjugates of pyrrole carboxylate), and the ether extract contained a prominent peak of radioactivity eluting before pyrrole carboxylate (Fig. 3A) ; These findings suggested that after pyrrole carboxylate administration, urinary pyrrole carboxylate is largely present as conjugates and other metabolites but that urinary pyrrole carboxylate formed from administered allohydroxy-n-proline is almost entirely free pyrrole carboxylate. The data noted above also imply a significant conversion of allohydroxy-n-proline to products other than pyrrole carboxylate.
Excretion of Pyrrole Carboxylate in Alkalinized Urine-The findings above suggested that pyrrole carboxylate formed in uiuo from hydroxy-n-proline or allohydroxy-n-proline, via Al-pyrroline-4-hydroxy-2-carboxylate, might arise by dehydration of the excreted pyrroline compound in the urine rather than in the tissues of the animal; this would explain the extensive formation of pyrrole carboxylate derivatives when pyrrole carboxylate itself was administered to the rat, and their absence from the urine when pyrrole carboxylate was formed in uiuo from a metabolic precursor. The relative stability of the pyrroline compound in alkaline solution2 suggested an experiment in which rat urine was made alkaline by NaHC03 ingestion. If a significant fraction of pyrrole carboxylate were formed in the urine, then this process might be sufficiently slowed in alkaline urine to permit accumulation of the pyrroline compound itself, and its detection as hydroxyproline after NaBH4 reduction. In one such experiment, urine was collected every 2.5 hours for 58 hours from six rats; the urine pH varied between 7 and 8. At each 2.5-hour interval, half the urine collected was treated with NaBH+ as described under "Methods." Total pyrrole carboxylate in the untreated urine aliquots was 0.34 pmol, while the NaBHI-treated urine contained 0.15 pmol.
Eflect of Antibiotic Treatment-The possibility has been noted (9) that hydroxy-n-proline might be converted to pyrrole carboxylate by way of preliminary epimerization to allohydroxy-nproline in the rat intestine. In initial experiments outlined earlier (ll), pyrrole carboxylate excretion after a large hydroxy-n-proline load was uninfluenced by essentially complete removal of fecal bacteria, as tested by aerobic colony formation. The high level of pyrrole carboxylate excreted in response to a hydroxy-nproline load (average of 0.53 pmol/lOO g in a control period; average of 0.60 ~mol/lOO g after gut sterilization) obviated the objection that pyrrole carboxylate was measured by Method 1; at this high level of pyrrole carboxylate, Method 1 and Method 2 gave equivalent results (see above). However, in a further test of the influence of antibiotic treatment on endogenous pyrrole carboxylate excretion, Method 1 also was used initially. Although there was no apparent effect of antibiotic treatment on endogenous pyrrole carboxylate excretion, the nonspecificity of the method for pyrrole carboxylate might have concealed real differences. Accordingly, the experiment was repeated with six rats, which were injected intraperitoneally with hydroxy-L-[G-aH]proline (3.3 x lo* dpm as a total dose), both before and 3 days after beginning antibiotic treatment, when feces appeared sterile. Radioactivity in pyrrole carboxylate in both the pre-and posttreatment periods represented 0.05% of the dose, indicating no demonstrable effect of antibiotic treatment.

Studies with Tissue Preparations
Pyrrole Carboxylate Formation in Homogenates-In preliminary incubations of rat kidney and rat liver homogenates with L-proline or hydroxy-L-proline, hydroxy-L-proline appeared to be a far better source of pyrrole carboxylate; at 0.2 M hydroxy-L-proline, 800 to 900 nmol of apparent pyrrole carboxylate were formed per g wet weight of kidney per hour. The reaction mixture, treated with p-dimethylaminobenzaldehyde, formed a colored product whose spectrum between 450 and 600 nm was identical with that formed by authentic pyrrole carboxylate. Liver homogenates had about 25% the activity of kidney homogenates. In similar incubations with L-proline the formation of pyrrole carboxylate was not detectable. From the sensitivity of the calorimetric assay, less than 3 nmol of true pyrrole carboxylate per g of tissue per hour could have been formed from proline.
Stability of Pyrrole Carboxylate and of the Pyrroline Precursor of Pyrrole Carboxylate-The conversion of hydroxy-L-proline to pyrrole carboxylate in kidney homogenates implied the intermediate formation of Al-pyrroline-4-hydroxy-2-carboxylate, a known source of pyrrole carboxylate and a plausible oxidation product of hydroxy-L-proline.
Pyrrole carboxylate itself was shown to be stable in kidney homogenates by direct addition and incubation under the same conditions. It was also necessary to test the stability of Al-pyrroline-4-hydroxy-2-carboxylate, since pyrrole carboxylate might be only one among other products of this intermediate. Because hydroxy-L-proline is predominantly oxidized to Al-pyrroline-3-hydroxy-5-carboxylate in kidney preparations (9, lo), allohydroxy-n-proline was used as a more efficient precursor of the ketimine pyrroline product, via n-amino acid oxidase catalysis. Accordingly, 1 pmol of this substrate was incubated with 0.5 ml of rat kidney homogenate under the conditions noted above. After 3 hours at 37", assays showed the formation of 0.45 pmol of pyrrole carboxylate and the presence of 0.62 pmol of allohydroxy-n-proline.
Elimination of D-Amino Acid Oxidase as the Major Basis for Homogenate Activity-Pyrrole carboxylate formation from hydroxy-L-proline might have resulted from n-amino acid oxidase action on a contaminating n-hydroxyproline epimer. This was ruled out on two grounds. First, the hydroxy-cproline used was shown by tests with crystalline n-amino oxidase (19) to contain less than 1 part of a D epimer in 200,000 parts of the L isomer, while the pyrrole carboxylate formed sometimes exceeded 1 part per thousand of the hydroxy-L-proline.
Furthermore, most of the pyrrole carboxylate-forming activity in the homogenate was recovered in the supernatant fraction after centrifugation at 25,000 x g for 2 hours, while most of the n-amino acid oxidase activity (measured by pyrrole carboxylate formation from allohydroxy- a With L-leucine as substrate. b Polyacrylamide gel electrophoresis yielded three bands; the major band, estimated at 45yo of total protein by densitometry, corresponded to the enzyme activity. Approximately 30yo of both the L-leucine and hydroxy-L-proline-oxidizing activity applied to the gel was recovered from this region. The -fold purification is based on the estimated removal of non-enzyme protein. n-proline) remained in the pellet. From the ratio of oxidation rates of saturating hydroxy-L-proline to saturating n-alanine (based on crystalline hog kidney n-amino acid oxidase (19)), the n-amino acid oxidase present could have accounted for less than 10% of the pyrrole carboxylate-forming activity observed in incubations with hydroxy-L-proline.
Identijication of L-Amino Acid Oxidase as the Major Enzyme-The L-amino acid oxidase of rat kidney proved capable of oxidizing hydroxy-Lproline to yield pyrrole carboxylate, after acidification of the reaction mixture. Using L-leucine as substrate, the enzyme was purified as described earlier (23) to the final step before crystallization.
This procedure yielded material approximately SO-fold purified, while polyacrylamide gel electrophoresis provided a final homogeneous fraction, 180-fold purified, after elution from the gel. In all fractions tested, the ratio of rates with L-leucine and hydroxy-L-proline remained approximately constant (Table III). Both L-leucine-and hydroxy-L-proline-oxidizing activity were lost in parallel on heating a purified fraction (Fraction 8, Table III), for 10 min at several temperatures between 50 and 65", at which all activity was lost (2).
Kinetic Constants for Hydroxy-L-proline- Fig.  4 shows a reciprocal plot for the oxidation of hydroxy-L-proline and its inhibition by L-leucine, using purified enzyme. These data and comparable data for L-leucine as a substrate provided values shown in Table IV, which includes a K, value for L-proline in a cruder enzyme fraction.
Specific Activity of Recovered Pyrrole Carboxylate-It was reported elsewhere (15) that, in the preparation of [G-3H]pyrrole carboxylate from hydroxy-L-[G-3H]proline, 50% of the tritium is lost; 35% in the enzymatic epimerization to allohydroxy-n-proline and 15y0 in the oxidation of the latter to the pyrroline compound and subsequent dehydration to pyrrole carboxylate. The same over-all loss of tritium was measured in the conversion of hydroxy-L-[G-aH]proline to pyrrole carboxylate by the partly purified L-amino acid oxidase (Fraction 4, Table III). This result is consistent with the oxidative route via ketimine formation  Table III). A, no inhibitor; B, addition of 0.038 M L-leucine. Pyrrole carboxylate (PCA) formed was measured after a-hour incubation at 37". The enzyme was Fraction 8 (Table III). b Determined with a less purified preparation. K,,, values for L-leucine and hydroxy-L-proline with the same preparation were similar to those shown for the purified enzyme. catalyzed by L-amino acid oxidase, since this step would remove the same tritium (at carbon 2) as does hydroxyproline-2-epimerase (28). In contrast, after administering hydroxy [G-3H]proline to intact rats, the specific activity of urinary pyrrole carboxylate was twice that of urinary free hydroxyproline (Table  II) or four times that expected for this conversion, by the findings above.

Nonenzymatic
Oxidation of Hydroxy-L-proline to Pyrrole Carboxylate-Observations presented in detail elsewhere (2) revealed significant catalysis of hydroxy-n-proline oxidation to pyrrole carboxylate by boiled homogenates of various tissues, including rat, guinea pig, and human kidney. A variety of experiments suggested that this reaction was catalyzed by metals rather than by tissue flavins. Compared with the enzymatic reaction, the nonenzymatic reaction was relatively small in rat kidney homogenates, but was more prominent than the minimal enzymatic reaction in homogenates of human or guinea pig kidney (see below). Studies of its temperature dependence (2) suggested that the nonenzymatic reaction was not involved in the in tivo conversion of hydroxy-L-proline to pyrrole carboxylate in any of the species studied. Rats were injected subcutaneously with 300 mg of L-proline and killed with ether at the times shown. Methods for the determination of free proline and hydroxyproline are described under "Methods." Values were obtained from pooled livers of three rats (untreated group) or from pooled livers of two rats in each group following proline administration.
In separate experiments, rats given large loads of proline excreted greatly increased free hydroxyproline in urine collected for 6 hours following proline administration.
Minutes after proline injection Oxidase by L-Proline-One possible explanation for the slight stimulation of pyrrole carboxylate excretion by administration of proline loads (Fig. 1) is selective inhibition by proline of the major pathway of hydroxy-L-proline oxidation, i.e. oxidation to Ai-pyrroline-3-hydroxy-5-carboxylate (9,10). To investigate this, the effect of proline on tissue levels of hydroxy-n-proline was examined; in addition, direct inhibition by proline of crude hydroxy-n-proline oxidase was tested.
The data of Table V show that a large subcutaneously administered n-proline load decreased the free hydroxy-n-proline in liver. Direct tests showed that, at high concentrations, L-proline markedly inhibited formation of the major pyrroline oxidation product of hydroxy-L-proline (Table VI). Al-Pyrroline-3hydroxy-5carboxylate proved stable after addition to these incubation mixtures, as also may be inferred from the stoichiometric balance between added hydroxy-n-proline, hydroxy-n-proline remaining, and Ai-pyrroline-hydroxy-5-carboxylate formed (Table VI).

Pyrrole Carboxylate Formation and Excretion in Guinea Pigs
The reported absence of n-amino acid oxidase from guinea pig kidney (29) prompted an examination of guinea pig urine for its content of pyrrole carboxylate.
After subcutaneous administration of hydroxy-n- [5-3H]proline to male albino guinea pigs, excretion of radioactive pyrrole carboxylate (Method 1) was only one-fifth to one-tenth the comparably measured value in the rat, not correcting for the body weight differences. Endogenous excretion of unlabeled pyrrole carboxylate (Method 1) was only about one-fourth that measured comparably in rat urine (11). Because Method 1 later was shown to overestimate true pyrrole carboxylate both in rat urine and human urine, even these low values for guinea pig urine may be falsely high, and from our present data we cannot be certain that any pyrrole carboxylate is normally excreted in guinea pig urine.
In agreement with early assays (29)) we were unable to demonstrate unequivocal n-amino acid oxidase in homogenates of guinea pig kidney, either with n-leucine or hydroxy-r-proline as substrate; pyrrole carboxylate formation from hydroxy-n-proline was no greater than 5% that of rat kidney homogenates. This was not explained by instability of pyrrole carboxylate or of Al-pyrroline-4-hydroxy-2-carboxylate in guinea pig kidney as tested by additions to homogenates.

Observations with Human Kidney Preparations
In contrast with the indecisive data on pyrrole carboxylate excretion by guinea pigs, there is clear evidence for pyrrole carboxylate excretion by man (15). In preliminary results with human kidney preparations, n-leucine oxidation could not be demonstrated either with a homogenate or with a fraction purified through ammonium sulfate precipitation to the stage of Fraction 4, Table III. However, apparent formation of pyrrole carboxylate from 0.4 M hydroxy-n-proline was catalyzed by the ammonium sulfate fraction, and the activity was inhibited over 90% by 8 x lOma M benzoate. The fractions tested contained n-amino acid oxidase, as detected by allohydroxy-n-proline oxidation, but the quantity of hydroxy-n-proline converted to pyrrole carboxylate (0.003 To) exceeded the calculated contamination of hydroxyn-proline with a n-epimer. The low level of reaction (less than 1% that of a rat kidney homogenate) is compatible with catalysis of pyrrole carboxylate formation from hydroxy-n-proline by Damino acid oxidase (19) rather than by an n-amino acid oxidase of human kidney.

DISCUSSION
Present data support the earlier conclusion, based on more limited and partly erroneous measurements (II), that in the rat exogenous hydroxy-n-proline is a far better source of urinary pyrrole carboxylate than is exogenous n-proline. In man (15), similarly, urinary pyrrole carboxylate was elevated either by an oral hydroxy-n-proline load or in congenital hydroxyprolinemia. In the rat, radioactive hydroxy-L-proline was a more efficient source of pyrrole carboxylate than was radioactive proline by 2 orders of magnitude, a difference which would appear to exceed the bias of diluting the labeled precursor into a larger body pool (Table V) .
Further questions concern the site and mechanism of hydroxyn-proline conversion to pyrrole carboxylate. Our findings, summarized in part earlier (ll), and subsequent data provided by Iguchi et al. (12), implicate the n-amino acid oxidase of rat kidney. The observations detailed here conclusively extend earlier find-ings in that the homogeneous enzyme was shown to act on hydroxy-n-proline; additionally, in crude kidney homogenates the n-amino acid oxidase reaction accounted for most of the relevant oxidizing activity. This question was raised by our finding, reported separately (19), that mammalian kidney n-amino acid oxidase also catalyzes a slow oxidation of hydroxy-n-proline to the same pyrroline product. Perhaps surprisingly, in view of the many examples in which intestinal flora account for trace metabolic conversions in mammals (30), effective sterilization of the gut had no influence either on the excretion of pyrrole carboxylate following a hydroxy-n-proline load, or on its endogenous production.
In connection with the above experiments, two findings remain unexplained. Administration of unlabeled n-proline resulted in a small (not more than a-fold), but seemingly definite, increase in urinary pyrrole carboxylate (Fig. 1). Possible explanations include stimulation of collagen turnover by a large proline load,3 as well as interference by high proline blood levels with the renal tubular reabsorption of pyrrole carboxylate, or of its precursor, Al-pyrroline-4-hydroxy-2-carboxylate.
We have investigated another possibility: that n-proline inhibits the main pathway oxidation of hydroxy-n-proline and hence makes the latter more accessible to the minor oxidative pathway via n-amino acid oxidase. Our findings were unexpected in that free hydroxy-n-proline of liver was decreased rather than increased, after a proline load.4 Experiments with kidney homogenates, however, did indicate marked inhibition of hydroxy-n-proline oxidase by high concentrations of n-proline, an observation not previously reported, to our knowledge. These findings, while of interest in themselves, did not provide an explanation for the stimulation of pyrrole carboxylate excretion by a proline load.
A second observation that we cannot now explain is the 3-to 4-fold higher specific radioactivity of urinary pyrrole carboxylate than that expected from the specific radioactivity of the free hydroxy-n-proline of urine, whether the source of label was L-[ U-%]proline or hydroxy-n-[G-3H]proline (Table II). It should be noted that, in contrast, the expected specific activity ratio was obtained in the pyrrole carboxylate formed from hydroxy-nproline by kidney homogenates. Since the same specific activity increment in pyrrole carboxylate, over that of the urinary hydroxyproline, resulted from administering either tritiated hydroxy-n-proline or carbon-labeled n-proline, both an isotope effect peculiar to tritium labeling, or a metabolic fate peculiar to proline seem doubtful as explanations. As an alternative we suggest possible time-or place-compartmentation of hydroxyproline oxidation (to the cyclic ketimine) relative to hydroxyproline excretion.
Another question concerns the site at which pyrrole carboxyla Several reports suggest an effect of L-proline concentration on collagen synthesis. One study involved direct measurements in isolated systems such as rat calvaria (31); others (32, 33) noted correlations between the size of the proline pool in liver and the amount of collagen or rate of collagen synthesis in hepatic fibrosis after tissue damage. The possibility that, in liver, both an elevated proline pool and fibrosis may be unrelated responses to tissue damage has also been pointed out (34).
4 Wolf and Berger (35) reported a la-fold increase in the free hydroxyproline of liver in rats fed large amounts of n-proline; increases in plasma hydroxyproline after proline administration both to rats (36) and man (37) have been described. These findings are uninterpretable in the absence of assurance that the large quantities of proline administered were free of significant contamination with hydroxyproline; hydroxpproline is known to be a significant contaminant of commercial samples of L-proline, unless steps are taken to purify the latter (38). ate is formed. Several findings favor the conclusion that, as a product of administered hydroxy-L-proline, pyrrole carboxylate is formed in the urine from the labile ketimine oxidation product of hydroxy-L-proline, Al-pyrroline-4-hydroxy-2-carboxylate. Thus, administered pyrrole carboxylate is converted almost entirely to urinary metabolic products, largely water-soluble, both in the present studies and in man (15). In contrast, when pyrrole carboxylate was formed in tivo from radioactive allohydroxy-nproline, no water-soluble radioactivity was detected in the urine other than residual allohydroxy-n-proline itself, and no radioactive peaks in the ether-soluble fraction of urine were found other than free pyrrole carboxylate (Fig. 3B). Alkalinization of rat urine, to stabilize the presumptive pyrroline precursor of endogenous origin, gave suggestive but not conclusive support to the postulation above: from these trials 56% of the pyrrole carboxylate could have been present in alkaline urine as the pyrroline precursor. These experiments would have appeared conclusive had none or all of the endogenously formed pyrrole carboxylate behaved as if derived from an alkali-stable, NaBHd-reducible precursor. The result obtained is consistent with the hypothesis examined if the relatively slow rate of dehydration of Ai-pyrroline-4-hydroxy-2carboxylate measured2 in simple solutions at pH 7 to 8 is accelerated by unknown components of urine. At present, our findings seem consistent with the thesis that much or all of the urinary pyrrole carboxylate, either of endogenous origin or after precursor administration, results from excretion of Al-pyrroline-4-hydroxy-2-carboxylate and its conversion to pyrrole carboxylate at some stage after the formation of urine.
While our data are consistent with endogenous free hydroxy-L-proline as the major, if not the only, source of urinary pyrrole carboxylate, a number of alternative hypothetical pathways cannot be excluded. These include possible formation of pyrrole carboxylate from 3,4-dehydroproline (39) (itself hypothetically derivable from L-proline) from the in wivo degradation of sialic acids, from y-hydroxyornithine (by transamination at the CYamino group (4)), or from 3-hydroxyproline. Certain of these possibilities appear unlikely from our data. Thus, radioactive n-proline, a likely precursor of 3,4-dehydroproline on a priori grounds, was not a significant precursor of pyrrole carboxylate beyond the rate compatible with conversion of n-proline to hydroxy-n-proline, nor did administration of unlabeled N-acetylneuraminate support the sialic acids as a physiological source (2). While y-hydroxyornithine might give rise, by (Y transamination, to the pyrroline precursor of pyrrole carboxylate, there is no evidence for the former compound in animal tissues. The role of L-amino acid oxidase in the formation of Al-pyrroline-4-hydroxy-2-carboxylate from hydroxy-L-proline is well supported by our studies with rats. The low, or ml, pyrrole carboxylate excretion by guinea pigs is consistent with our inability (and that reported earlier (29)) to demonstrate unambiguous n-amino acid oxidase in guinea pig kidney. Adult humans excrete approximately 18fold less pyrrole carboxylate, on a body weight basis, than do the immature rats studied in the present work; the estimated daily release of free hydroxy-n-proline from collagen in the two cases corresponds approximately to this ratio (15), and evidence like that cited for the rat also implicates hydroxy-n-proline as a source of pyrrole carboxylate in human urine (15). Yet, activity for hydroxy-L-proline oxidation to the cyclic ketimine was barely demonstrable in human kidney homogenates, so that in man other pathways for this reaction should be considered.
Certain of the observations reported here bear on the metabolic fate of allohydroxy-n-prohne or Al-pyrroline-4-hydroxy-Z-car-boxylate formed from it. In studies of kidney homogenates, we could demonstrate satisfactory balances, indicating that the pyrroline compound was stable under our incubation conditions and was the only product of allohydroxy-n-proline. However, results after administering allohydroxy-n-[G-3H]proline in vivo pointed to extensive metabolism either of allohydroxy-n-proline or its pyrroline product, since only 50% of the administered tritium was recovered. The possibility that the missing radioactivity had been diluted in body Hz0 was considered under "Results." A plausible product is 3-hydroxy-4aminobutyrate, earlier demonstrated to arise from Ai-pyrroline-4-hydroxy-2-carboxylate in in vitro experiments (4). As noted above, kidney homogenates did not catalyze formation of this product in our experiments, possibly because of the inclusion of catalase in incubation mixtures. A parenthetic point of interest was our failure to find radioactive hydroxy-n-proline in rat urine after administering allohydroxy-o-[G-3H]proline. This also argues against significant epimerization of hydroxyproline by gut bacteria in tivo (or reabsorption of the reaction product) and separately, implies that enzymatic reduction of A'-pyrroline-4-hydroxy-2-carboxylate to hydroxy-L-proline is not a significant reaction in the rat; the corresponding conversion of Al-pyrroline-2-carboxylate to L-proline was observed in several rat tissues (40) * Finally, the possible application of our findings merits some comment. The reaction we have studied would appear to represent a new pathway (although a minor one) for the metabolism of hydroxy-L-proline in mammals. XIeasurement of urinary pyrrole carboxylate therefore provides a possible index of collagen turnover which might respond differently to various alterations of collagen metabolism than do other indices such as urinary hydroxyproline (41). As yet we have no information under this heading, and ready accumulation of data would need a simpler assay for free pyrrole carboxylate than the one we have developed.
Some preliminary findings of ours are worth noting. Although most of the studies in this paper were based on immature male rats, a few observations with female rats of the same age and strain indicated no sex difference, by the relatively undiscriminating procedure of Method 1. Administration to rats of triiodothyronine (100 pg/lOO g of body weight) led to no significant increase in urinary pyrrole carboxylate or in the apparent activity of kidney L-amino acid oxidase. Urinary pyrrole carboxylat,e in this case also was measured by the earlier method, which includes pyrrole carboxylate-unrelated components; however, any prominent increase in true pyrrole carboxylate excretion would have been detected. Our findings in this respect do not support the conclusion of Yaminishi et al. (14) that pyrrole carboxylate is increased in human hyperthyroidism.