Defective hydroxyproline metabolism in type II hyperprolinemia

DIOCHEMICAL

MEDICINE

Defective

10,

329-336

( 1974)

Hydroxyproline Metabolism Hyperprolinemia’

in Type II

STEPHEN I. GOODMAN, JOHN W. MACE, BARBARA S. MILES, CECILIA C. TENG, AND SUZANNE B. BROWN3 Department of Pediatrics, Uniuersity of Colorado Medical Center, 4200 East Ninth Auenue, Denuer, Colorado 80220 Received

October

23,

1973

The specific purpose of this paper is to report upon investigations in Type II hyperprolinemia which indicate that there is a block in the metabolism of both hydroxyproline and proline in this disorder. Early steps in the parallel metabolism of L-proline and 4-hydroxy-Lproline are shown in Fig. 1. Both are metabolized through Al-pyrroline derivatives; the carbon skeleton of proline entering the tricarboxylic acid cycle as cy-ketoglutarate while that of hydroxyproline is metabolized through pyruvate and glyoxylate. Proline oxidase and hydroxyproline oxidase appear to be different enzymes in man; the evidence for this rests mainly upon the observation that hydroxyproline oxidase deficiency and hyperhydroxyprolinemia could not be demonstrated in a patient with hyperprolinemia due to documented deficiency of proline oxidase ( 1). It is not yet clear whether the reduction of Al-pyrroline-5-carboxylic acid and of Al-pyrroline-3-hydroxy-5-carboxylic acid is catalyzed by an identical enzyme or distinct enzymes ( 2, 3) ; it is similarly uncertain whether Al-pyrroline-5-carboxylic acid dehydrogenase is identical with or distinct from Al-pyrroline-3-hydroxy-5-carboxylic acid dehydrogenase (4, 5). Two types of hyperprolinemia have been described in man (6) ; Type I has been shown to be due to deficiency of proline oxidase (1) while Type II is presumed due to deficiency of A’-pyrroline-5-carboxylic acid * Supported in part by a Centers Program of the Division by NIH Grant HD-04024 and ’ Department of Pediatrics, California. ’ Department of Pediatrics, que, New Mexico. Copyright All rights

Grant (RR-69) f rom the General Clinical Research of Research Services, National Institutes of Health, by Maternal and Child Health Special Project 252. Loma Linda University Medical Center, Loma Linda, University

of New

329 0 1974 by Academic Press, Inc. of reproduction in any form reserved.

Mexico

Medical

Center,

Albuquer-

3:30

GOODMAN

ET

AL.

:cOOII PROLINE

&PYRROLINE. -5-CARBOXYLIC ACID

GLUTAMIC

ACID

(1”: <‘=
YoH :~“(~H?’ ‘c’ ” (:tI-oH o~coo~~c~~~~,+ yz - p, < Gf$Lfc H

4-HYDROXYPROLINE

AX)”

<:ooH

:.=o (!OOJl

~~-PYRROLINE. 3-HYDROXY. 5-CARBOxyLiC ACID

7-HYDBOXYGLUTAMIC ACID

I-HYDROXY. s-KETOGLUTARIC ACID

PYRUVIC ACID

FIG. 1. Early steps in the catabolism of L-proline and 4-hydroxy-L-proline. Proline oxidase. @ Hydroxyproline oxidase. @ A’-pyrroline-5-carboxylic acid hydrogenase. @ A’-pyrroline-5-carboxylic acid reductase. @ A’-pyrroline-3-hydroxy5-carboxylic acid reductase. The fact that there is a common A’-pyrroline-5-carboxylic acid dehydrogenase has not been firmly established (4, 5), nor is it certain that reductases are separate and distinct enzymes (2, 3).

@ de-

the

dehydrogenase. Both conditions arc characterized by hyperprolinemia and iminoglycinuria, the latter being due to saturation by proline of an active transport system in the renal tubule which shares affinity for proline, hydroxyproline, and glycine (7). In addition, however, the urine in Type II hyperprolinemia reacts positively with o-aminobenzaldehyde, presumably because of the presence of Al-pyrroline-5-carboxylic acid, and it has been this reaction which has led to the tentative placement of the metabolic block. The results presented in this paper indicate that both A’-pyrroline-5carboxylic acid and A’-pyrroline-%hydro,uy-S-carboxylic acid are excreted in the urine in this disease, that the latter is the prime o-aminobenzaldehyde positive compound, and that there is a defect in the catabolism of although a deficiency of hydroxyproline in these patients. Moreover, Al-pyrroline-5-carboxylic acid dehydrogenase is compatible with all of the observed biochemical abnormalities, a primary defect at the level of proline oxidase cannot be ruled out except by direct enzyme assay. CASE

SUMMARY

The patient is an 11-yr-old Spanish-American girl who to the Mental Development Center at the University of School of Medicine at 9 yr of age because of her mother’s her “lack of motivation.” Evaluation at that time showed

was brought New Mexico concern over normal intel-

TYPE

II

HYPERPROLINEMIA

331

lectual function (IQ = 88; W.I.S.C.), the parents were reassured, and the child has since done well in a normal school setting. There is no history of seizures, and physical examination is completely normal. Unidimensional amino acid chromatography of her urine in butanol: acetic acid : water ( 12 : 3 : 5) showed iminoglycinuria and the urine gave a positive reaction with o-aminobenzaldehyde ( 1). Ion-exchange chromatography confirmed the iminoglycinuria and, in addition, revealed the presence of two unusual compounds whose identities were established as detailed below. The child’s parents are not related, and none of her three siblings have either iminoglycinuria or hyperprolinemia.

-Methods Urine samples were refrigerated after voiding, and were pooled and frozen at -20” at the end of each 24-hr collection period. Plasma samples were frozen at - 20”. Concentrations of amino acids in plasma and urine were determined using a Beckman model 120 automatic acid analyzer. Plasma proteins were precipitated with sulfosalicylic acid, 67 mg/ml, immediately prior to analysis. For oral 4-hydroxyproline loading, the L-isomer was dissolved in fruit juice and administered in amounts of 100 mg/kg body wt. al-Pyrroline-3-hydroxy-5-carboxylic acid was prepared by rabbit kidney catalyzed conversion from 4-hydroxy-L-proline ( 8). It reacted positively with o-aminobenzaldehyde (Sigma Chemical Company) and was eluted from a 55-cm column of Beckman UR-30 Custon Spherical Resin (30” ) by 0.2 M sodium citrate buffer, pH 3.26, just after 4hydroxyproline. The 5701440 nm absorbance ratio of its complex with citrate and ninhydrin was 3.36. Al-Pyrroline-5-carboxylic acid was synthesized as described by Strecker (9); the y,y-dicarbethoxy-y-acetamidobutyraldehyde phenylhydrazone required was synthesized from ethylacetamidomalonate ( 10) purchased from Nutritional Biochemicals Corp. The synthetic compound reacted positively with o-aminobenzaldehyde and was eluted from UR-30 as a single peak just after glutamine-asparagine. The 570/440 nm absorbance ratio of its complex with citrate and ninhydrin was 0.65. RESULTS

1. Urine and Pla.sm.u Amino Acids Urine amino acids were normal except for marked iminoglycinuria and two abnormal compounds which were eluted from UR-30 resin by 0.2 M sodium citrate, pH 3.26, as shown in Fig. 2. Peak 1 was the larger of the

332

Elutlon

Time -I”

Minutes

FIG. 2. Part of ion-exchange chromatogram of urine amino acids; solid line = absorbance at 570 nm, broken line = absorbance at 440 ML Peak 1 has been identified as due to A.‘-pyrroline-3-hydroxy-5-carboxylic acid; peak 2 has been tentatively identified as due to A’-pyrroline-5-carboxylic acid.

two, was present in all urines examined, was eluted just after li-hydroxyproline, and had a 5701440 nm absorbance ratio of 3.68. Peak 2 was smaller and only variably present, was eluted just after glutamineasparagine, and absorbed more at 440 nm than at Tj70 nm. When serial fractions were collected from the bottom of the resin column, only the fraction containing peak 1 reacted positively with o-aminobenzaldehyde. The concentrations of free amino acids in plasma (fasting) were normal except for proline, which varied from 1.658 to 2.534 pmoles/ml (normal = 0.070-0.150 pmoles/ml), and minor elevations as follows. Hydroxyproline concentration was 0.021 pmoles/ml (normal < 0.01) and the concentrations of alanine and ornithine were approximately twice normal. A small 440 nm peak corresponding in location to urine peak 2 was noted only once; no other abnormal compounds were detected. At a plasma proline concentration of 2.471 ,umoles/ml, the clearances of hydroxyproline, proline, and glycine were 27.5, 5.6, and 13.8%of the glomerular filtration rate (endogenous creatinine clearance), respectively. In the normal subject, clearances of proline and hydroxyproline are less than l%, and that of glycine less than 6%,of the glomerular filtration rate. 2. Identification of Abnormal Compounds (a) A’-Pyrroline-3-hydroxy-S-carboxylic acid, Peak 1 cochromatographed with, and had similar 5701440 nm absorbance properties as the compound, presumably A’-pyrroline-3-hydroxy-S-carboxylic acid, (a)

TYPE

II

HYPERPROLINEMIA

333

which was synthesized from 4-hydroxy-L-proline by rabbit kidney, and (b) which appeared in the urine of control subjects after hydroxyproline loading ( see below). The positive reaction with o-aminobenzaldehyde confirmed that the abnormal compound was C=N containing (11). 4Hydroxyproline was the sole product of sodium borohydride reduction (8) of the urine fraction containing it; in particular, no allo-hydroxyproline was formed, indicating that the compound was not Al-pyrroline4-hydroxy-2-carboxylic acid. Sodium borohydride reduction of the latter produces approximately equimolar amounts of hydroxy+proline and allohydroxy-n-proline ( 12). Also weighing against the compound’s identity as Al-pyrroline-4-hydroxy-2-carboxylic acid is its stability; the latter is quite labile (13) and readily becomes dehydrated to form pyrrole-2-carboxylic acid, an Ehrlich-positive compound which was never noted on paper chromatograms of the patient’s urine. Quantitative reduction to hydroxyproline by sodium borohydride provided a factor which was then used to estimate A’-pyrroline-3-hydroxyS-carboxylic acid concentrations in plasma and urine of the patient and controls. (b) A’-Pyrroline-s-carborylic acid. Peak 2 was eluted with and had similar 570/440 nm absorbance properties as synthetic Al-pyrroline-5carboxylic acid. A positive reaction with o-aminobenzaldehyde could not be obtained with the quantities present in the patient’s urine. 3. Hydroryproline

Loading

Figure 3 shows plasma hydroxyproline istration of this amino acid (100 mglkg)

FIG. 3. Plasma hydroxyproline concentrations 4-hydroxy-L-praline to the patient (solid line) broken line with open circles shows results praline oxidase deficiency ( 14 ) .

concentrations after adminto the patient and to two age-

following oral loads of 100 mg/kg and two controls (broken lines). The obtained on a patient with hydroxy-

Before load

O-2.;, hl

L’.T,-G hr

Hydroxyproline-patient, cant rol A control B

1.148 0 0

42.713

29.712

~1-Pyrroline-Y-OH-5-C00H patient) control A control B

0.438 0 0

.01.5

0

,073

0

3 038 0.011 0.004

3 ,250 0 0

matched controls. Also indicated are levels observed under the same conditions in a patient with proven hydroxyproline oxidase deficiency (14). The peak concentration achieved by the patient with hyperprolinemia Type II was greater than that observed both in the controls and in hydroxyprolinemia. A compound with the elution and absorbance properties of A’pyrroline-Shydroxy-Scarboxylic acid was detected in small amounts in the plasma of the patient after loading; it was not detected in the control subjects. Table 1 shows the urinary excretion of hydroxyproline and .L’pyrroline-3-hydroxyScarboxylic acid in the patient and controls before and after loading; increases in the excretion of these compounds were much more marked in the patient. DISCUSSION

The child described in this communication shows the characteristic biochemical phenotype of Type II hyperprolinemia, that is, marked hyperprolinemia, iminoglycinuria, and a positive urine reaction with oaminobenzaldehyde. Ion-exchange chromatography of urine revealed the same abnormalities as those reported previously in another patient with this disorder (15). The results presented above indicate that the predominant o-aminobenzaldehyde positive compound excreted in this disease is not A’pyrroline-5-carboxylic acid but Al-pyrroline-3-hydroxy-5-carboxylic acid, an intermediate in the metabolism of 4-hydroxy-L-proline. This compound was identified on the basis of chromatographic properties as well as its reactions with ninhydrin, o-aminobenzaldehyde, and sodium borohydride. A block in the metabolism of hydroxyproline was further sug-

TYPE

II

HYPERPROLINEMIA

335

gested by (a) the moderate fasting hyperhydroxyprolinemia and (b) the results of hydroxyproline loading, particularly the high plasma hydroxyproline levels produced and the urinary excretion of such large amounts of hydroxyproline and Al-pyrroline-3-hydroxy-5carboxylic acid. Thus, there appears to be a block in the metabolism of both proline and hydroxyproline in this disorder. While the biochemical findings are compatible with a block at the level of a common Al-pyrroline-S-carboxylic acid dehydrogenase, the presence of separate and distinct enzymes for A’-pyrroline-5-carboxylic acid and A’-pyrroline-3-hydroxy-5carboxylic acid cannot be ruled out. For instance, a block at the level of the hydroxypyrroline dehydrogenase might cause accumulation of Alpyrroline-3-hydroxy-5-carboxylic acid which might then inhibit Alpyrroline-5carboxylic acid dehydrogenase and thus lead to accumulation of its substrate. Given a block at this level, i.e., A’-pyrroline-5-carboxylic acid dehydrogenase( s ), a plausible explanation for the discrepancy between the magnitudes of elevation of serum proline and hydroxyproline may be provided by the properties of the Al-pyrroline reductase system. In beef liver (2), the K, for Al-pyrroline-5-carboxylic acid is roughly l/loo that for A’-pyrroline-3-hydroxy-5carboxylic acid (4.9 X 1O-s M vs 4.4 X lo-” M) , and V,,,,, with the former is two to three times that with the latter. Such properties favor more rapid and efficient reduction to proline than to hydroxyproline. If the properties of the human Al-pyrroline reductase system are similar, its action might convert a situation in which both A’-pyrroline compounds were accumulating to one characterized in the main by hyperprolinemia and A’-pyrroline-3-hydroxy-5-carboxylic aciduria. Moreover, while the biochemical abnormalities in this disorder are compatible with a block at the level of A*-pyrroline-5-carboxylic acid dehydrogenase, it cannot be stated with certainty that the block is due to a mutation at this point. For instance, proline is a potent inhibitor of beef A’-pyrroline-5carboxylic acid dehydrogenase (4, 5)) inhibiting activity by approximately 65% at 3.3 X lo-” M. As this concentration is present in the plasma of many patients with this disorder, the primary defect in the disorder might be a severe K,?,,mutation of proline oxidase with (a) synthesis of Al-pyrroline-5-carboxylic acid at high intracellular proline concentrations, and (b) secondary inhibition of Al-pyrroline-5carboxylic acid dehydrogenase. While direct assay of the enzymes of proline metabolism will be necessary to settle this point, we believe that secondary inhibition by proline is unlikely since some, albeit few, patients with proline oxidase deficiency (1, 16) have had plasma proline levels as high or higher than some patients with Type II hyperprolinemia ( 15, 17)) and have not had o-aminobenzaldehyde-positive urines.

336

(:C)OI)I\IAS

ET

AL..

Seizures and mental retard;hon have: been noted in most previous patients with this disorder ( 1, 1.5, 18 ) but ~vcre not present in this child, may have been due to suggesting perhaps that previous associations sample bias. Indeed, an 8-yr-old affected sibling of the proband reported upon by Simila ( 18) was clinically normal, a finding in accord with this hypothesis. SUMMARY Studies are reported on a patient with Type II hyperprolinemia. The results indicate that there is a defect in the metabolism of both proline and hydroxyproline in this disorder, and that the prime o-aminobenzaldehyde positive compound in the urine of these patients is A’-pyrrolineof either Al-pyrroline-s3-hydroxy-5-carboxylic acid. Deficiencies carboxylic acid dehydrogenase or proline oxidase are compatible with the biochemical phenotype, and direct enzyme assays will be necessary to settle this point. ACKNOWLEDGMENTS We thank Dr. J. Albert Browder, Mrs. Nancy Weaver, and Mrs. Sharon Pecha of the Mental Development Center, University of New Mexico School of Medicine, for the assistance given us during this study. REFERENCES 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

EFRON, M. L., N. Engl. J. hfed. 272, 1243 (1965). ADAMS, E., AND GOLDSTONE, A., J. Biol. Chem. 235, 3499 (1960). PEISACH, J., AND STRECKEH, H. J., J. Biol. Chem. 237, 2255 (1962). STRECKER, H. J., J. Biol. Chem. 235, 3218 (1960). ADAMS, E., AND GOLDSTONE, A., J. Biol. Chem. 23.5, 3504 (1960). SCHIVER, C. R., AND EFHON, M. L., in “The Metabolic Basis of Inherited Disease” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, Eds.), 3rd ed., p. 351. McGraw-Hill Book Co., New York, 1972. SCRIVER, C. R., EFRON, M. L., AND SCHAFER, I. ,4., J. Clin. Invest. 43, 374 (1964). ADAMS, E., AND GOLDSTONE, A., J. Biol. Chem. 23.5, 3492 (1960). STRECKEH, H. J., J. Biol. Chem. 235, 2045 (1960). MOE, 0. A., AND WARNER, D. T., J. Amer. Chem. Sot. 70, 2763 (1948). STHECKEH, H. J., J. Biol. Chem. 225, 825 ( 1957). SINGH, R. M. M., AND ADAMS, E., J. Biol. Chem. 240, 4344 (1965). RADHAKHISHNAN, A. N., AND MEISTEH, A., J. Biol. Chem. 226, 559 (1957). EFHON, M. L., BIXBY, E. ht., AND PRYLES, C. V., N. Engl. J. Med. 272, 1299 (1965). E~~EHY, F. A., GOLDIE, L., AND STERN, J., J. Ment. Dcf. Res. 12, 187 (1968). PIESOWICZ, A. T., Arch. Dis. Child. 43, 748 (1968). JEUNE, M., COLLAMBEL, C., MICHEL, M., DAVID, H., GUIBAUD, P., GUERFUIH, G., AND ALBERT, J., Ann. Pediat. 46, 349 ( 1970). SIhfILX, S., Ann. Chn. Bes. 2, 143 ( 1970).