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Bound hydroxyproline excretion following gelatin loading in prolidase deficiency
Bound Hydroxyproline Excretion Following Loading in Prolidase Deficiency
Gelatin
Gerald F. Powell and Rose Mary Maniscaico The excretion of peptide-bound hydroxyproiine before and after gelatin loading was evaluated in two children with prolidase deficiency, two adult heterozygotes, and normal controls. On a low hydroxy proline diet, the patients with prolidase deficiency excreted 6.9 and 2.4 times more bound hydroxyproline than normal children. The bound hydroxyproline excretion for the heterozygotes was comparable to the adult controls. Children ingested 20 g of gelatin and adults 29 g. In the 24 hr following gelatin loading, the homozygotes excreted 14.4 and 17.3 times more of the ingested load of hydroxyproline than did normal children. This constituted 39% and 47% of
the hydroxyproline ingested. Of the hydroxyproline excreted in 24 hr, 58%, and 61.4% was excreted in the first 6 hr. Over the 24 hr period, the normal children excreted 2.7% of the hydroxyproline ingested (97.8% in the first 6 hr). The heterozygotes excreted only slightly more than the adult controls. The normal adults excreted 6.0% of the ingested hydroxyproline (82.2% in the first 6 hr). In prolidase deficiency, large amounts of peptide-bound hydroxyproline can cross the intestinal wall unhydrolyzed. Prolidase appears to have an important role in normal hydrolysis of peptide-bound proline.
hydroxy-
C
OLLAGEN AND ELASTIN are the only animal proteins containing Hydroxyproline constitutes significant amounts of hydroxyproline. all hydroxyproline in pep13%-14x of collagen and 2% of elastin. ’ Essentially tide form or free hydroxyproline is derived from the degradation of collagen. Hydroxyproline is synthesized by hydroxylation of selected proline residues already in peptide linkage in a precursor of collagen. Because of the unique relationship of peptide-bound hydroxyproline to collagen and because a specific chemical assay is available to measure free hydroxyproline, urinary peptidebound hydroxyproline excretion has been used as an index of collagen metabolism. Intestinal absorption of hydroxyproline peptides in adults has been clearly demonstrated by the measurement of urinary bound hydroxyproline after the ingestion of gelatin.2A Up to 8% of the hydroxyproline present in the ingested gelatin is excreted in the peptide-bound form. How much bound hydroxyproline is actually absorbed and subsequently metabolized rather than excreted as the peptide is unknown. Several investigations5-’ have suggested a relative resistance of gelatin peptides to intestinal hydrolysis. After feeding gelatin to rats, Rogers, et al.6 demonstrated that more nitrogen is recovered from the intestine at timed intervals
From the Departments of Pediatrics and Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston. Tex. This work was supported by Grant RR-73 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. Reprint requests should be addressed to Gerald F. Powell, M.D.. Associate Professor, Department of Pediatrics, The University of Texas Medical Branch, Galveston, Tex. 77550. :s 1976 bv Grune & Stratton, Inc.
Metabolism,
Vol. 25, No. 5 (May), 1976
503
504
POWELL
AND MANISCALCO
than when the rats are fed casein or egg albumin. Similar findings were demonstrated in man.’ It has been suggested’ that the epithelial cells of the small intestine may lack an enzyme or enzymes capable of splitting the specific amino acid linkages present in gelatin peptides. Of the amino acids naturally occurring in proteins only proline and hydroxyproline have an (Yamino nitrogen within a ring structure and therefore but a single hydrogen on the nitrogen. They are thus designated as imino acids. When these two imino acids are in the C-terminal position of a dipeptide, the peptide linkage formed is an imide bond. Prolidase is the only enzyme known to specifically cleave imidodipeptides. Because of its high content of proline and hydroxyproline, one-quarter of the peptide bonds of collagen are imido bonds. For this reason, prolidase is an important enzyme in completely degrading collagen to free amino acids. Prolidase is present in human intestinal mucosa* and probably plays an important role in hydrolysis of imidodipeptides prior to absorption into the portal blood. Recently a patient has been described with a deficiency of prolidase and a tenfold increase in excretion of bound hydroxyproline as compared to normal children.’ The excretion of peptide-bound hydroxyproline following gelatin loading in two patients with prolidase deficiency and two heterozygotes is the subject of this report. The study was undertaken to determine if exogenous sources of bound hydroxyproline contribute to the increased excretion of bound hydroxyproline and to investigate the role of prolidase in intestinal cleavage of peptide-bound hydroxyproline. MATERIALS
AND METHODS
Two unrelated patients with prolidase deficiency and their mothers were studied. Four normal adults (two male and two female) and four normal children, ages 8-12 yr, served as controls. The I2 yr old, a female, was in early adolescence, the others were prepubertal. The adults are faculty members and the parents of the children studied. One of the patients with prolidase deficiency has been previously reported’ as the first documented example of prolidase deficiency in man. The second patient, a female, as yet unreported has similar clinical findings and prolidase deficiency in white cells and serum. The clinical features common to both subjects include frequent and chronic upper respiratory infections, chronic dermatitis, and splenomegaly, as well as massive imidodipeptiduria. The obligate heterozygotes studied are clinically normal and have approximately 50% of the normal adults mean white blood cell prolidase activity (Table I). Day I served as a control for day 2 on which the subjects received a gelatin load. After fasting over night, the subjects continued fasting from 600 a.m. to l2:OO noon on day I. From 12:OO noon on, on both days, the subjects were on a normal diet except for avoidance of gelatin containing foods. After fasting over night, at 6:00 a.m., on day 2, the children ingested 20 g of commercial Table 1. White Blood Cell Prolidars Activity (pmoles Dipeptide Hydrolyzed/mg Patient TK Mother PK Patient SR Mother BR
Protein/Hour) 0.00 1.48 0.00 1.91
Children (N = 6)
2.65 f 0.13(2.47-2.79)*
Adults (N = 8)
3.17 f 0.37(2.74-3.77)
*Mean f SD (range).
BOUND
gelatin
HYDROXYPROUNE
and
the
adults
505
EXCRETION
29
g.
They otherwise
fasted until
noon.
The gelatin
water heavily flavored with orange Tang, and ingested while still warm. 14% hydroxyproline
based on the dry weight.
adults is comparable
to that given to adults in studies by other
report of a gelatin load in children”
The 29 g of gelatin
was dissolved
The gelatin
selected for ingestion
investigators.2A.‘0.”
I4 g were given. A slightly
larger
in hot
used contained In
by the
the only
load was selected for this
study. Urine was collected for the entire 24-hr period on both days but in consecutive lections starting at 6:00 a.m. Free hydroxyproline Loxley.‘2
Total
urinary
hydroxyproline
was determined
was determined
after
by the method
a l6-hr
I IO’C. The difference between the free and total hydroxyproline
hydrolysis
6 and I8 hr colof Bergman
and
in 6 N HCI
at
is the bound hydroxyproline.
RESULTS
The excretion of bound hydroxyproline is seen in Table 2. On day 1, the total 24-hr excretion of bound hydroxyproline for the normals (adults and children) excreted more than adults. is comparable to published data. 13-16 Children Hydroxyproline excretion in the heterozygotes (PK and BR) was within the normal adult range. The 24-hr urinary hydroxyproline excretion for TK, one of the patients, is 6.9 times the mean for normal children. That for SR is 2.4 times the mean for normal children. The 6-hr excretion for SR is within the normal range for children while that for TK is considerably above that. Evaluation of the fasting data suggest that patient TK has (1) a greater endogenous collagen turnover than SR, (2) a more severe enzyme deficiency, or perhaps both. The increased excretion of bound hydroxyproline in the two patients most likely results from decreased hydrolysis of peptide bound hydroxyproline because of the deficiency of prolidase. Excretion of bound hydroxyproline following gelatin loading is seen on the right hand side of Table 2. The total 24-hr excretion in the adults is comparable to that in the literature2e3 following ingestion of similar amounts of gelatin. The excretion by the heterozygotes is within the range for adult normals. Comparable data for children following gelatin loading is not available from the literature. Excretion of bound hydroxyproline by the patients is 10.1 and 8.7 times the mean for normal children. Table 2. mg/6 hr
Bound Hydroxyproline
mg/l8
hr
Excretion*
mg/24 hr totalt
(mg)
mg/6 hr
mg/l8
hr
mgJ24 hr total7
Norm&$ 10.9
Adults
zt4.1 19.4
Children
zt8.9
f
36.1
47.0
14.5
zt17.8
183.0 *41
07.7
68.3 zt24.1
+31.4
73.3
256.3
+21.4
zt45.3
83.1
68.7
152.8
k-36.2
zt22.0
zt57.8
Homorygotes
TK
190.2
409.8
608.0
740.0
803.3
1544.1
SR
27.7
183.8
211.5
718.7
610.0
1336.7
Heterozygotes PK
7.1
23.4
30.5
228.2
BR
11.6
36.7
48.3
159.3
*Bound periods
hydroxyproline
excretion
on each of two
consecutive
t The 24-hr $The
normal
column values
(total
hydroxyproline
days.
is the sum of the 6 and we
the mean of four
Consecutive
minus 6 and
free
and four
hydroxyproiine)
18 hr collections
18 hr collections. adults
children.
79.4
307.6
114.1
273.4 in mg for
were carried
out.
specific
time
506
POWELL
AND
MANISCALCO
Table 3. Bound Hydroxyproline Excretion Following Gelatin loading B*f%\
A’(w)
C’(%i
Normals Adults
209.3t
6.0
82.2
65.1
2.7
97.8
Children Homozygotes TK
936.1
38.9
58.0
SR
1125.2
46.7
61.4
PK
277.1
7.9
79.8
8R
225.1
6.4
65.6
Heterozygotes
*A
Bound
day 2 minus *8
Bound
droxyproline
hydroxyproline total
mg/24
hydroxyproline ingested.
(mg)
hr, day
excreted
excreted
(Children
in
24
hr
as
a
result
of
gelatin
loading.
(Total
mg/24
hr.
1). per
24
ingested
hr
2408
expressed mg
as
a
percentage
hydroxyproline-adults
of
the
actual
ingested
3490
amount
of
mg
hydroxy-
hy-
proline.) *C creted
Bound
hydroxyproline
excreted
during
1st
6
hr
in Table
1.
expressed
OS a
percentage
of
hydroxyproline
ex-
in 24 hr.
tcalculated
from
the mean of the normals
Bound hydroxyproline excreted as a direct result of gelatin loading is seen in Table 3. In column A, the 24-hr bound hydroxyproline (mg) listed, is the difference between that excreted on day 1 (control) and that excreted on day 2 (gelatin load). The homozygotes excreted, respectively, 14 and 17 times more of the ingested load of hydroxyproline than did normal children in 24 hr. Both heterozygotes excreted slightly more than the normal adults. The quantity of bound hydroxyproline excreted in 24 hr expressed as a percentage of hydroxyproline ingested is seen in column B of Table 3. The subjects with prolidase deficiency excreted 39% and 47% of the total hydroxyproline ingested. From the data, it can be interpreted that in prolidase deficiency, a large amount of bound hydroxyproline can be absorbed across the intestinal wall without being hydrolyzed and that a large percentage of the peptides absorbed are not hydrolyzed by other tissues prior to excretion. The heterozygotes excreted 7.9% and 6.4% of the hydroxyproline ingested which is similar to the mean adult normal. The normal children excreted much less than the normal adults despite greater hydroxyproline intake per kilogram. Of the total mg of bound hydroxyproline excreted in 24 hr the per cent excreted in the first 6 hr is seen in column C of Table 3. The controls excreted more than 80% of the peptide hydroxyproline in the first 6 hr. The subjects with prolidase deficiency and one of the heterozygotes excreted less than two-thirds of the ingested hydroxyproline during the 6-hr period. Since there is no known mechanism for renal reabsorption of bound hydroxyproline, the decrease in percentage excreted during the first 6-hr period in the subjects as compared to the normal may suggest delayed intestinal absorption of bound hydroxyproline. DISCUSSION
The process of intestinal absorption of small peptides involves two mechanisms,i7vishydrolysis of peptides by brush border enzymes with subsequent cellular uptake of the liberated amino acids, and uptake of peptide by mechanisms
BOUND
HYDROXYPROUNE
EXCRETION
507
independent of specific amino acid transport systems, followed by intracellular hydrolysis. The independence of mucosal uptake of amino acids and peptides has been demonstrated by studies of Gly (Gly-Pro) influx in rabbit ileal mucosai9 and by absorption of amino acids in peptide form that are not absorbed in the free form in subjects with Hartnup disease2’v2’ and cystinuria.22*23 For the most part, hydrolysis of the peptide is completed in the intestinal wall. There is little absorption of peptide into the portal blood, although transmural absorption of small amounts of peptide has been reported. Hydroxyproline peptides appear in the peripheral plasma and urine in man after feeding of gelatin.24v24 The major peptide excreted following gelatin loading is Pro-Hyp.25 A similar excretion of hydroxyproline peptides occurs in rats and hamsters.24 With small intestine in vitro, transmural transport of Pro-Hyp in the hamste? and transmural transport of Pro-Hyp, Gly-Pro, Pro-Gly, Pro-Pro, and .27 With the exception of Pro-Gly, Leu-Pro in the rat have been demonstrated these dipeptides require prolidase for cleavage to free amino acids. Boullin et a1.28 reported that intact Gly-Pro and Pro-Gly as well as carnosine, Gly-Gly, Gly- 1-Phe, and Gly-D-Phe can be detected in the superior mesentary vein after intraluminal injection of dipeptides into anesthetized rats and that there was an apparent inverse relationship between the rate of hydrolysis and detection of the intact peptide. The extent to which dipeptides can be absorbed into portal blood in man is unknown. Excretion of bound hydroxyproline following gelatin loading demonstrates that at least 87: of the bound hydroxyproline ingested can cross the intestinal wall unhydrolyzed. The amount of bound of hydroxyproline excreted may not reflect that amount absorbed in peptide form since hydrolysis of the peptide after transmural transport is possible in tissues other than the intestine. The bound hydroxyproline excretion following gelatin loading in prolidase deficiency demonstrates that in the absence of intracellular hydrolysis large amounts of peptide bound hydroxyproline can cross the intestinal wall. Assuming a relatively complete deficiency of prolidase in these subjects, the amount of peptide bound hydroxyproline absorbed would be reflected in the amount excreted since hydrolysis by other tissues would not occur. These studies in prolidase deficiency suggest that prolidase plays a role in normal metabolism, both in regard to the amount of peptide hydroxyproline absorbed and the rate of absorption of these dipeptides. In the gelatin loading studies referred to as well as the present one, the assay for peptide hydroxyproline is not specific for dipeptide and larger peptides could be included in the determination of bound hydroxyproline. However, prolidase is specific for dipeptides and does not cleave tripeptides. This suggests that the increased bound hydroxyproline excretion in prolidase deficiency is in the dipeptide form. This study indicates that bound hydroxyproline excreted in this disorder may have an exogenous source as well as an endogenous source. Prolidase activity present in the heterozygotes is sufficient to handle a gelatin load in a near normal fashion. The decreased excretion of bound hydroxyproline following gelatin loading in normal children as compared to normal adults may possibly be due to greater activity of prolidase in children as compared to adults.
508
POWELL
AND MANISCALCO
REFERENCES 1. Neuman RE. Logan MA: The determination of collagen and elastin in tissues. J Biol Chem 186549, 1950 2. Bronstein HD, OD: The significance
Haetliner LJ, Kowlessar of gelatin tolerance in
malabsorption states. Gastroenterology 50:621, 1966 3. Prockop DJ, Sjoerdsma A: Significance of urinary hydroxyproline in man. J Clin Invest 40:843, 1961 4. Prockop DJ, Keiser H, Sjoerdsma A: Gastrointestinal absorption and renal excretion of hydroxyproline peptides. Lancet 2:527, 1962 5. Chen ML, Rogers QR, Harper AE: Observations on protein digestion In Vivo IV. Further observations on the gastrointestinal contents of rats fed different dieting proteins. J Nutrition 76:235, 1962 6. Rogers QR, Chen ML, Peraino C, Harper AE: Observations on protein digestion in vivo. 111.Recovery of nitrogen from the stomach and small intestine at intervals after feeding diets containing different proteins. J Nutrition 72: 331, 1960 7. Nixon SE, Mawer GI: The digestion and absorption of protein in man. I. The site of absorption. Br J Nutr 24:227, 1970 8. Heizer WD, Laster L: Peptide hydrolase activities of the mucosa of human small intestine. J Clin Invest 48:210, 1969 9. Powell GF, Rasco MA, Maniscalco RM: A prolidase deficiency in man with iminopeptiduria. Metabolism 23:505, 1974 IO. Theil GB, Benoit FL, Watten RH: An oral gelatin-xylose test for estimating pancreatic proteolytic activity. Am J Digest Dis 8:1008. 1963 Il. Cerda JJ, Brooks FP, Prockop DJ: Intraduodenal hydrolysis of gelatin as a measure of protein digestion in normal subjects and in patients with malabsorption syndromes. Gastroenterology 54:358, 1968 12. Bergman 1, Loxley R: The determination of hydroxyproline in urine hydrolysates. Clin Chim Acta 27:346, 1970 13. Laitinen 0, Nikkila EA. Kivirikko KI: Hydroxyproline in the serum and urine. Normal values and clinical significance. Acta Med Stand 179:275, 1966 14. Benoit FL, Theil GB, Watten RH: Hy-
droxyproline excretion in endocrine disease. Metabolism 12: 1072. 1963 15. Jasin HE, Fink CW, Wise W, Ziff M: Relationship between urinary hydroxyproline and growth. J Clin Invest 41:1928, 1962 16. Kivirikko Kl, Laitinen 0: Clinical significance of urinary hydroxyproline determination in children. Ann Paediat Fenn 11:148. 1965 17. Silk DBA: Progress report. Peptide absorption in man. Gut 15:494, 1974 18. Matthews DM: Intestinal absorption of peptides. Physiol Review 55:537, 1975 19. Rubino A, Field M, Shwachman H: Intestinal transport of amino acid residues of dipeptides. J Biol Chem 246:3542, 1971 20. Asatoor AM, Cheng B, Edwards KDG, Lant AF, Matthews DM, Milne MD, Navab F, Richards AJ: intestinal absorption of two dipeptides in Hartnup disease. Gut 11:380, 1970 21. Navab F, Asatoor AM: Studies on intestinal absorption of amino acids and a dipeptide in a case of Hartnup disease. Gut ll:373, 1970 22. Hellier MD, Holdsworth CD, Perrett D, Thirumalai C: Intestinal dipeptide transport in normal and cystinuric subjects. Clin Sci 43: 659, 1972 23. Hellier MD, Perrett D, Holdsworth CD: Dipeptide absorption in cystinuria. Br Med J 4:782, 1970 24. Hueckel HJ, Rogers QR: Urinary excretion of hydroxyproline-containing peptides in man, rat. hamster, dog and monkey after feeding gelatin. Comp Biochem Physiol 32:7, 1970 25. Meilman E, Urivetzky CM: Urinary hydroxyproline Invest 42:40, 1963
MM, Rapoport peptides. J Clin
26. Hueckel HJ, Rogers QR: Prolylhydroxyproline absorption in hamsters. Can J Biochem 501782, 1972 27. Saidel LJ, Edelstein I: Hydrolysis and absorption of proline dipeptides across the wall of sacs prepared from everted rat intestine. Biochem Biophys Acta 367:76, 1974 28. Boullin DJ, Crampton RF, Pelling D: Intestinal absorption containing glycine, phenylalanine, alanine or histidine in the rat. Med 45:849, 1973
Heading CE, of dipeptides proline, betaClin Sci Mel
Gelatin
Gerald F. Powell and Rose Mary Maniscaico The excretion of peptide-bound hydroxyproiine before and after gelatin loading was evaluated in two children with prolidase deficiency, two adult heterozygotes, and normal controls. On a low hydroxy proline diet, the patients with prolidase deficiency excreted 6.9 and 2.4 times more bound hydroxyproline than normal children. The bound hydroxyproline excretion for the heterozygotes was comparable to the adult controls. Children ingested 20 g of gelatin and adults 29 g. In the 24 hr following gelatin loading, the homozygotes excreted 14.4 and 17.3 times more of the ingested load of hydroxyproline than did normal children. This constituted 39% and 47% of
the hydroxyproline ingested. Of the hydroxyproline excreted in 24 hr, 58%, and 61.4% was excreted in the first 6 hr. Over the 24 hr period, the normal children excreted 2.7% of the hydroxyproline ingested (97.8% in the first 6 hr). The heterozygotes excreted only slightly more than the adult controls. The normal adults excreted 6.0% of the ingested hydroxyproline (82.2% in the first 6 hr). In prolidase deficiency, large amounts of peptide-bound hydroxyproline can cross the intestinal wall unhydrolyzed. Prolidase appears to have an important role in normal hydrolysis of peptide-bound proline.
hydroxy-
C
OLLAGEN AND ELASTIN are the only animal proteins containing Hydroxyproline constitutes significant amounts of hydroxyproline. all hydroxyproline in pep13%-14x of collagen and 2% of elastin. ’ Essentially tide form or free hydroxyproline is derived from the degradation of collagen. Hydroxyproline is synthesized by hydroxylation of selected proline residues already in peptide linkage in a precursor of collagen. Because of the unique relationship of peptide-bound hydroxyproline to collagen and because a specific chemical assay is available to measure free hydroxyproline, urinary peptidebound hydroxyproline excretion has been used as an index of collagen metabolism. Intestinal absorption of hydroxyproline peptides in adults has been clearly demonstrated by the measurement of urinary bound hydroxyproline after the ingestion of gelatin.2A Up to 8% of the hydroxyproline present in the ingested gelatin is excreted in the peptide-bound form. How much bound hydroxyproline is actually absorbed and subsequently metabolized rather than excreted as the peptide is unknown. Several investigations5-’ have suggested a relative resistance of gelatin peptides to intestinal hydrolysis. After feeding gelatin to rats, Rogers, et al.6 demonstrated that more nitrogen is recovered from the intestine at timed intervals
From the Departments of Pediatrics and Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston. Tex. This work was supported by Grant RR-73 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. Reprint requests should be addressed to Gerald F. Powell, M.D.. Associate Professor, Department of Pediatrics, The University of Texas Medical Branch, Galveston, Tex. 77550. :s 1976 bv Grune & Stratton, Inc.
Metabolism,
Vol. 25, No. 5 (May), 1976
503
504
POWELL
AND MANISCALCO
than when the rats are fed casein or egg albumin. Similar findings were demonstrated in man.’ It has been suggested’ that the epithelial cells of the small intestine may lack an enzyme or enzymes capable of splitting the specific amino acid linkages present in gelatin peptides. Of the amino acids naturally occurring in proteins only proline and hydroxyproline have an (Yamino nitrogen within a ring structure and therefore but a single hydrogen on the nitrogen. They are thus designated as imino acids. When these two imino acids are in the C-terminal position of a dipeptide, the peptide linkage formed is an imide bond. Prolidase is the only enzyme known to specifically cleave imidodipeptides. Because of its high content of proline and hydroxyproline, one-quarter of the peptide bonds of collagen are imido bonds. For this reason, prolidase is an important enzyme in completely degrading collagen to free amino acids. Prolidase is present in human intestinal mucosa* and probably plays an important role in hydrolysis of imidodipeptides prior to absorption into the portal blood. Recently a patient has been described with a deficiency of prolidase and a tenfold increase in excretion of bound hydroxyproline as compared to normal children.’ The excretion of peptide-bound hydroxyproline following gelatin loading in two patients with prolidase deficiency and two heterozygotes is the subject of this report. The study was undertaken to determine if exogenous sources of bound hydroxyproline contribute to the increased excretion of bound hydroxyproline and to investigate the role of prolidase in intestinal cleavage of peptide-bound hydroxyproline. MATERIALS
AND METHODS
Two unrelated patients with prolidase deficiency and their mothers were studied. Four normal adults (two male and two female) and four normal children, ages 8-12 yr, served as controls. The I2 yr old, a female, was in early adolescence, the others were prepubertal. The adults are faculty members and the parents of the children studied. One of the patients with prolidase deficiency has been previously reported’ as the first documented example of prolidase deficiency in man. The second patient, a female, as yet unreported has similar clinical findings and prolidase deficiency in white cells and serum. The clinical features common to both subjects include frequent and chronic upper respiratory infections, chronic dermatitis, and splenomegaly, as well as massive imidodipeptiduria. The obligate heterozygotes studied are clinically normal and have approximately 50% of the normal adults mean white blood cell prolidase activity (Table I). Day I served as a control for day 2 on which the subjects received a gelatin load. After fasting over night, the subjects continued fasting from 600 a.m. to l2:OO noon on day I. From 12:OO noon on, on both days, the subjects were on a normal diet except for avoidance of gelatin containing foods. After fasting over night, at 6:00 a.m., on day 2, the children ingested 20 g of commercial Table 1. White Blood Cell Prolidars Activity (pmoles Dipeptide Hydrolyzed/mg Patient TK Mother PK Patient SR Mother BR
Protein/Hour) 0.00 1.48 0.00 1.91
Children (N = 6)
2.65 f 0.13(2.47-2.79)*
Adults (N = 8)
3.17 f 0.37(2.74-3.77)
*Mean f SD (range).
BOUND
gelatin
HYDROXYPROUNE
and
the
adults
505
EXCRETION
29
g.
They otherwise
fasted until
noon.
The gelatin
water heavily flavored with orange Tang, and ingested while still warm. 14% hydroxyproline
based on the dry weight.
adults is comparable
to that given to adults in studies by other
report of a gelatin load in children”
The 29 g of gelatin
was dissolved
The gelatin
selected for ingestion
investigators.2A.‘0.”
I4 g were given. A slightly
larger
in hot
used contained In
by the
the only
load was selected for this
study. Urine was collected for the entire 24-hr period on both days but in consecutive lections starting at 6:00 a.m. Free hydroxyproline Loxley.‘2
Total
urinary
hydroxyproline
was determined
was determined
after
by the method
a l6-hr
I IO’C. The difference between the free and total hydroxyproline
hydrolysis
6 and I8 hr colof Bergman
and
in 6 N HCI
at
is the bound hydroxyproline.
RESULTS
The excretion of bound hydroxyproline is seen in Table 2. On day 1, the total 24-hr excretion of bound hydroxyproline for the normals (adults and children) excreted more than adults. is comparable to published data. 13-16 Children Hydroxyproline excretion in the heterozygotes (PK and BR) was within the normal adult range. The 24-hr urinary hydroxyproline excretion for TK, one of the patients, is 6.9 times the mean for normal children. That for SR is 2.4 times the mean for normal children. The 6-hr excretion for SR is within the normal range for children while that for TK is considerably above that. Evaluation of the fasting data suggest that patient TK has (1) a greater endogenous collagen turnover than SR, (2) a more severe enzyme deficiency, or perhaps both. The increased excretion of bound hydroxyproline in the two patients most likely results from decreased hydrolysis of peptide bound hydroxyproline because of the deficiency of prolidase. Excretion of bound hydroxyproline following gelatin loading is seen on the right hand side of Table 2. The total 24-hr excretion in the adults is comparable to that in the literature2e3 following ingestion of similar amounts of gelatin. The excretion by the heterozygotes is within the range for adult normals. Comparable data for children following gelatin loading is not available from the literature. Excretion of bound hydroxyproline by the patients is 10.1 and 8.7 times the mean for normal children. Table 2. mg/6 hr
Bound Hydroxyproline
mg/l8
hr
Excretion*
mg/24 hr totalt
(mg)
mg/6 hr
mg/l8
hr
mgJ24 hr total7
Norm&$ 10.9
Adults
zt4.1 19.4
Children
zt8.9
f
36.1
47.0
14.5
zt17.8
183.0 *41
07.7
68.3 zt24.1
+31.4
73.3
256.3
+21.4
zt45.3
83.1
68.7
152.8
k-36.2
zt22.0
zt57.8
Homorygotes
TK
190.2
409.8
608.0
740.0
803.3
1544.1
SR
27.7
183.8
211.5
718.7
610.0
1336.7
Heterozygotes PK
7.1
23.4
30.5
228.2
BR
11.6
36.7
48.3
159.3
*Bound periods
hydroxyproline
excretion
on each of two
consecutive
t The 24-hr $The
normal
column values
(total
hydroxyproline
days.
is the sum of the 6 and we
the mean of four
Consecutive
minus 6 and
free
and four
hydroxyproiine)
18 hr collections
18 hr collections. adults
children.
79.4
307.6
114.1
273.4 in mg for
were carried
out.
specific
time
506
POWELL
AND
MANISCALCO
Table 3. Bound Hydroxyproline Excretion Following Gelatin loading B*f%\
A’(w)
C’(%i
Normals Adults
209.3t
6.0
82.2
65.1
2.7
97.8
Children Homozygotes TK
936.1
38.9
58.0
SR
1125.2
46.7
61.4
PK
277.1
7.9
79.8
8R
225.1
6.4
65.6
Heterozygotes
*A
Bound
day 2 minus *8
Bound
droxyproline
hydroxyproline total
mg/24
hydroxyproline ingested.
(mg)
hr, day
excreted
excreted
(Children
in
24
hr
as
a
result
of
gelatin
loading.
(Total
mg/24
hr.
1). per
24
ingested
hr
2408
expressed mg
as
a
percentage
hydroxyproline-adults
of
the
actual
ingested
3490
amount
of
mg
hydroxy-
hy-
proline.) *C creted
Bound
hydroxyproline
excreted
during
1st
6
hr
in Table
1.
expressed
OS a
percentage
of
hydroxyproline
ex-
in 24 hr.
tcalculated
from
the mean of the normals
Bound hydroxyproline excreted as a direct result of gelatin loading is seen in Table 3. In column A, the 24-hr bound hydroxyproline (mg) listed, is the difference between that excreted on day 1 (control) and that excreted on day 2 (gelatin load). The homozygotes excreted, respectively, 14 and 17 times more of the ingested load of hydroxyproline than did normal children in 24 hr. Both heterozygotes excreted slightly more than the normal adults. The quantity of bound hydroxyproline excreted in 24 hr expressed as a percentage of hydroxyproline ingested is seen in column B of Table 3. The subjects with prolidase deficiency excreted 39% and 47% of the total hydroxyproline ingested. From the data, it can be interpreted that in prolidase deficiency, a large amount of bound hydroxyproline can be absorbed across the intestinal wall without being hydrolyzed and that a large percentage of the peptides absorbed are not hydrolyzed by other tissues prior to excretion. The heterozygotes excreted 7.9% and 6.4% of the hydroxyproline ingested which is similar to the mean adult normal. The normal children excreted much less than the normal adults despite greater hydroxyproline intake per kilogram. Of the total mg of bound hydroxyproline excreted in 24 hr the per cent excreted in the first 6 hr is seen in column C of Table 3. The controls excreted more than 80% of the peptide hydroxyproline in the first 6 hr. The subjects with prolidase deficiency and one of the heterozygotes excreted less than two-thirds of the ingested hydroxyproline during the 6-hr period. Since there is no known mechanism for renal reabsorption of bound hydroxyproline, the decrease in percentage excreted during the first 6-hr period in the subjects as compared to the normal may suggest delayed intestinal absorption of bound hydroxyproline. DISCUSSION
The process of intestinal absorption of small peptides involves two mechanisms,i7vishydrolysis of peptides by brush border enzymes with subsequent cellular uptake of the liberated amino acids, and uptake of peptide by mechanisms
BOUND
HYDROXYPROUNE
EXCRETION
507
independent of specific amino acid transport systems, followed by intracellular hydrolysis. The independence of mucosal uptake of amino acids and peptides has been demonstrated by studies of Gly (Gly-Pro) influx in rabbit ileal mucosai9 and by absorption of amino acids in peptide form that are not absorbed in the free form in subjects with Hartnup disease2’v2’ and cystinuria.22*23 For the most part, hydrolysis of the peptide is completed in the intestinal wall. There is little absorption of peptide into the portal blood, although transmural absorption of small amounts of peptide has been reported. Hydroxyproline peptides appear in the peripheral plasma and urine in man after feeding of gelatin.24v24 The major peptide excreted following gelatin loading is Pro-Hyp.25 A similar excretion of hydroxyproline peptides occurs in rats and hamsters.24 With small intestine in vitro, transmural transport of Pro-Hyp in the hamste? and transmural transport of Pro-Hyp, Gly-Pro, Pro-Gly, Pro-Pro, and .27 With the exception of Pro-Gly, Leu-Pro in the rat have been demonstrated these dipeptides require prolidase for cleavage to free amino acids. Boullin et a1.28 reported that intact Gly-Pro and Pro-Gly as well as carnosine, Gly-Gly, Gly- 1-Phe, and Gly-D-Phe can be detected in the superior mesentary vein after intraluminal injection of dipeptides into anesthetized rats and that there was an apparent inverse relationship between the rate of hydrolysis and detection of the intact peptide. The extent to which dipeptides can be absorbed into portal blood in man is unknown. Excretion of bound hydroxyproline following gelatin loading demonstrates that at least 87: of the bound hydroxyproline ingested can cross the intestinal wall unhydrolyzed. The amount of bound of hydroxyproline excreted may not reflect that amount absorbed in peptide form since hydrolysis of the peptide after transmural transport is possible in tissues other than the intestine. The bound hydroxyproline excretion following gelatin loading in prolidase deficiency demonstrates that in the absence of intracellular hydrolysis large amounts of peptide bound hydroxyproline can cross the intestinal wall. Assuming a relatively complete deficiency of prolidase in these subjects, the amount of peptide bound hydroxyproline absorbed would be reflected in the amount excreted since hydrolysis by other tissues would not occur. These studies in prolidase deficiency suggest that prolidase plays a role in normal metabolism, both in regard to the amount of peptide hydroxyproline absorbed and the rate of absorption of these dipeptides. In the gelatin loading studies referred to as well as the present one, the assay for peptide hydroxyproline is not specific for dipeptide and larger peptides could be included in the determination of bound hydroxyproline. However, prolidase is specific for dipeptides and does not cleave tripeptides. This suggests that the increased bound hydroxyproline excretion in prolidase deficiency is in the dipeptide form. This study indicates that bound hydroxyproline excreted in this disorder may have an exogenous source as well as an endogenous source. Prolidase activity present in the heterozygotes is sufficient to handle a gelatin load in a near normal fashion. The decreased excretion of bound hydroxyproline following gelatin loading in normal children as compared to normal adults may possibly be due to greater activity of prolidase in children as compared to adults.
508
POWELL
AND MANISCALCO
REFERENCES 1. Neuman RE. Logan MA: The determination of collagen and elastin in tissues. J Biol Chem 186549, 1950 2. Bronstein HD, OD: The significance
Haetliner LJ, Kowlessar of gelatin tolerance in
malabsorption states. Gastroenterology 50:621, 1966 3. Prockop DJ, Sjoerdsma A: Significance of urinary hydroxyproline in man. J Clin Invest 40:843, 1961 4. Prockop DJ, Keiser H, Sjoerdsma A: Gastrointestinal absorption and renal excretion of hydroxyproline peptides. Lancet 2:527, 1962 5. Chen ML, Rogers QR, Harper AE: Observations on protein digestion In Vivo IV. Further observations on the gastrointestinal contents of rats fed different dieting proteins. J Nutrition 76:235, 1962 6. Rogers QR, Chen ML, Peraino C, Harper AE: Observations on protein digestion in vivo. 111.Recovery of nitrogen from the stomach and small intestine at intervals after feeding diets containing different proteins. J Nutrition 72: 331, 1960 7. Nixon SE, Mawer GI: The digestion and absorption of protein in man. I. The site of absorption. Br J Nutr 24:227, 1970 8. Heizer WD, Laster L: Peptide hydrolase activities of the mucosa of human small intestine. J Clin Invest 48:210, 1969 9. Powell GF, Rasco MA, Maniscalco RM: A prolidase deficiency in man with iminopeptiduria. Metabolism 23:505, 1974 IO. Theil GB, Benoit FL, Watten RH: An oral gelatin-xylose test for estimating pancreatic proteolytic activity. Am J Digest Dis 8:1008. 1963 Il. Cerda JJ, Brooks FP, Prockop DJ: Intraduodenal hydrolysis of gelatin as a measure of protein digestion in normal subjects and in patients with malabsorption syndromes. Gastroenterology 54:358, 1968 12. Bergman 1, Loxley R: The determination of hydroxyproline in urine hydrolysates. Clin Chim Acta 27:346, 1970 13. Laitinen 0, Nikkila EA. Kivirikko KI: Hydroxyproline in the serum and urine. Normal values and clinical significance. Acta Med Stand 179:275, 1966 14. Benoit FL, Theil GB, Watten RH: Hy-
droxyproline excretion in endocrine disease. Metabolism 12: 1072. 1963 15. Jasin HE, Fink CW, Wise W, Ziff M: Relationship between urinary hydroxyproline and growth. J Clin Invest 41:1928, 1962 16. Kivirikko Kl, Laitinen 0: Clinical significance of urinary hydroxyproline determination in children. Ann Paediat Fenn 11:148. 1965 17. Silk DBA: Progress report. Peptide absorption in man. Gut 15:494, 1974 18. Matthews DM: Intestinal absorption of peptides. Physiol Review 55:537, 1975 19. Rubino A, Field M, Shwachman H: Intestinal transport of amino acid residues of dipeptides. J Biol Chem 246:3542, 1971 20. Asatoor AM, Cheng B, Edwards KDG, Lant AF, Matthews DM, Milne MD, Navab F, Richards AJ: intestinal absorption of two dipeptides in Hartnup disease. Gut 11:380, 1970 21. Navab F, Asatoor AM: Studies on intestinal absorption of amino acids and a dipeptide in a case of Hartnup disease. Gut ll:373, 1970 22. Hellier MD, Holdsworth CD, Perrett D, Thirumalai C: Intestinal dipeptide transport in normal and cystinuric subjects. Clin Sci 43: 659, 1972 23. Hellier MD, Perrett D, Holdsworth CD: Dipeptide absorption in cystinuria. Br Med J 4:782, 1970 24. Hueckel HJ, Rogers QR: Urinary excretion of hydroxyproline-containing peptides in man, rat. hamster, dog and monkey after feeding gelatin. Comp Biochem Physiol 32:7, 1970 25. Meilman E, Urivetzky CM: Urinary hydroxyproline Invest 42:40, 1963
MM, Rapoport peptides. J Clin
26. Hueckel HJ, Rogers QR: Prolylhydroxyproline absorption in hamsters. Can J Biochem 501782, 1972 27. Saidel LJ, Edelstein I: Hydrolysis and absorption of proline dipeptides across the wall of sacs prepared from everted rat intestine. Biochem Biophys Acta 367:76, 1974 28. Boullin DJ, Crampton RF, Pelling D: Intestinal absorption containing glycine, phenylalanine, alanine or histidine in the rat. Med 45:849, 1973
Heading CE, of dipeptides proline, betaClin Sci Mel