Urinary peptide hydroxyproline before and during postpartum involution of human uterus

Urinary Peptide Hydroxyproline Before and During Postpartum Involution of Human Uterus B.YLEROY

KLEIN AND SAMUEL S. C. YEN

The metabolic fate of collagen during the involution of the uterus was studied by sequential analysis of urinary peptide hydroxyproline in 6 pregnant women during the second half of pregnancy and puerperium. The pattern of increasing levels of urinary peptide hydroxyproline during pregnancy and decreasing levels during the early puerperium suggests that involution of the uterus has little effect on the excretion of peptide hydroxyproline. Similar studies of a patient with massive intravascular hemolysis demonstrated that peptide hydroxyproline is

decreased but not totally destroyed in the presence of large amounts of erythrocyte prolidase, a specific peptidase for the hydrolysis of the major peptide hydroxyproline linkage. Thus, there is no known way for peptide hydroxyproline to escape undetected from the body, since peptide hydroxyproline cannot be metabolized to carbon dioxide. It is suggested that the collagen is resorbed from the uterus in a manner during postnon-destructive partum involution. (Metabolism 19: No. 1, January, 19-23, 1969)

HE POSTPARTUM INVOLUTION of the uterus represents one of nature’s experiments for studying the massive breakdown of muscle and connective tissue proteins under physiological conditions. It has been demonstrated”,” that during the first 8 to I I days postpartum, 22-53 Cm. of collagen (equivalent to 3-7 Cm. of hydroxyproline) are resorbed from the human uterus. The mechanism of collagen resorption:! and the metabolic fate of resorbed collagen have not been elucidated.” Although mammalian collagenases4 have been found in the rat uterus using tests in vitro, it is not known whether such destructive enzymes are operating in vivo under physiological conditions. If destruction of uterine collagen is occurring, there would be complete breakdown of collagen to the level of peptides, amino acids and carbon dioxide with their eventual excretion. It has been demonstrated” that approximately 20 per cent of degraded collagen must appear in the urine as peptide hydroxyproline because the prolyl-hydroxyproline linkage is relatively inert to enzymatic attack by the specific peptidase, prolidase. Without hydrolyzing peptides to free amino acids. thcrc cannot be any metabolic degradation of peptides to carbon dioxide,” their ultimate fate being excretion or reutilization.

T

bfI.T4HOLIShI.

VOL.

19, No.

1 (JANCIARY),

1970

I0

20

KLEIN AND YEN Table

Patient

AS

E.J. A.M. L.B. M.M. M.A. E.H.

_____

1.-Summary

of Clinical

and Urinary

Parity

Weeks of Gestation

Fetal Weight (Gm.)

1 1 4 5 7 7

36 40 40 37 38 40

2400 3180 2920 2710 2850 3660

21 23 23 29 30 35 -___ Table 2 .-Effect

Period

Control Crisis Crisis Resolving

Day

1 4 29

of Intravascular

Blood Hg~;~l;;qin Hematocrit (ml. per 100 ml.) 100 ml. )

11.8 9.5 6.1 11.0

* Per cent free = (free hypro/free

38 31 20 35

Hemolysis

66 51 30 57 37 33

50 32 31 35 24 26

-

on Urinary Hydroxyproline

Urinary Peptide (mg./.24 hr.)

Data

Urinary Peptide Hydroxyproline (mg. per 24 hr.) 5-l Weeks At delivery Postpartum

Hydroxyproline Free-(mg./24

64.0 35.0 27.2 60.0

hypro + peptide hypro)

hr.)

2.0 6.8 13.2 30.0

Urinary Free (as per cent Creatinine of total)* (m&24 hr.)

3 16 33 33

2060 1940 1950 1810

X 100.

To determine the metabolic fate of collagen, we have measured serially urinary peptide hydroxyproline during pregnancy and puerperium. In addition, a patient with acute intravascular hemolysis due to paroxysmal nocturnal hemoglobinuria was studied to quantitate the destructive effect of a massive release of erythrocyte prolidase on the levels of peptide hydroxyproline. METHODS Six pregnant patients were studied serially from the 20th week of pregnancy to the eighth week postpartum (Table 1). All appeared to be in good health prior to pregnancy, and had no complications during pregnancy, labor and puerperium. All were judged to have normal renal function as defined by blood-urea-nitrogen and creatinine clearance. The patients were placed on a gelatin-free diet one day before and during th 24-hour urine collections. Urinary creatinine levels were used to determine the completeness of the COIlections. Free and total hydroxyproline in urine were assayed as previously described.’ The difference between total and free hydroxyproline was assumed to represent peptide-bound hydroxyproline. A 60-year-old male patient with a history of repeated attacks of acute hemolysis, anemia, and hemoglobinuria was diagnosed as having paroxysmal nocturnal hemoglobinuria (Table 2). His erythrocytes were susceptable to hemolysis in acidified serum. There was no previous evidence of renal or- liver dysfunction. RESULTS

All of the six pregnant patients showed a gradual increase in urinary peptide hydroxyproline during the latter half of pregnancy (Fig. I ). Five of the six pregnant patients demonstrated decreasing levels of urinary peptide hydroxyproline during the puerperium, and one patient showed no change (Fig. 1). Free hydroxyproline varied 2-10 per cent of the total hydroxyproline (free plus peptide) during pregnancy and l-3 per cent during the puerperium. The period

?I

URINARYPEPTIDEHYDROXYPROLINE

z3

I I

., 24

.I.,

20

.,

.I.,

16

12

8

4

PREPARTUM

.I’,

0

4

0

POSTPARTUM WEEKS

Fig. l.-Excretion of urinary peptide hydroxyproline during pregnancy puerperium. Shaded area represents normal range of urinary hydroxyproline; vertical (broken) line is at parturition.

and and

of maximal excretion of peptide hydroxyproline occurred at 15-28 days before delivery for four patients, and at two days after delivery for two patients. The greatest decrease of urinary peptide hydroxyproline was observed in those patients that had the largest increase during the third trimester of pregnancy. Three of the six patients (M.A., E.H., E.J.) began to decrease their level of peptide hydroxyproline two to three weeks prior to delivery. With the exception of patient E.J., the levels of urinary peptide hydroxyproline by the 6th postpartum week were at the same level as those observed at mid-pregnancy. The patient with paroxysmal nocturnal hemoglobinuria demonstrated a dccreased level of urinary peptide hydroxyproline and an increased level of urinary free hydroxyproline during an acute hemolytic episode (Table 2 1. This resulted in a 58 per cent decrease in peptide hydroxyproline as compared to the control value. Twenty-live days after the hemolytic crisis, the urinary peptide hydroxyproline had returned to the previous baseline value but the free hydroxyproline had remained elevated. DISCUSSION

The increased level of urinary peptide hydroxyproline during pregnancy and its rate of decrease during the puerperium appear to be a reflection of the prcgnant state rather than the involution of the uterus. Meilman et al.x have observed marked decreases in the daily levels of urinary peptide hydroxyproline during 3-5 days postpartum in two subjects. Previous preliminary studies:’ in three patients had demonstrated that the total urinary hydroxyproline reached its maximal level during 2-6 days postpartum. In contrast to the hydroxyproline excretion, high levels of urinary nitrogen

22

KLEIN

AND YEN

are excreted for the first 2-12 days postpartum” with a maxima1 excretion occurring between 5-X days postpartum. I” The nitrogen balance is negative for almost two weeks.!’ Montfort and Perez-Tamayo l1 have demonstrated that there is an equivalent loss of muscle protein and collagen during the involution. Thus, the involuting tissues are not being physically lost from the body via vaginal discharge of lochia, or the gastrointestinal tract.“.” Serum polysaccharide’:’ and serum mucoprotein I-I levels reach a maximum between 6-10 days postpartum followed by a gradual decrease over several weeks. The urine and blood observations plus the changes in uterine weight and collagen’.” are consistent with a massive involution of the uterus occurring by the end of the first week postpartum. The lack of increase in urinary peptide hydroxyproline during the same period suggests that uterine collagen is not being proteolytically destroyed by acid cathepsin15 or collagenase.‘” An alternative explanation is that the uterus contains all the necessary peptidases so that collagen would be completely hydrolyzed to amino acids which would be metabolized to carbon dioxide by the liver without peptide hydroxyproline appearing in the urine. This catabolism would occur if the uterus contains large amounts of prolidase which would hydrolyze the prolyl-hydroxyproline linkage (which represents 60-75 per cent of the urinary peptide hydroxyproline).” Smith et al.” have emphasized that prolidase is present in low concentrations in most tissues, the lowest concentration being found in the human uterus and serum.‘” The highest levels of prolidase are found in kidney, intestine, and erythrocytes. Since prolidases from the different tissues have the same substrate specificity in their action,” varying specificities of enzymatic action can be eliminated from consideration. Since human erythrocytes are impermeable to peptides,l!’ a patient with intravascular hemolysis was studied because the circulating hydroxyproline peptides would be exposed to a massive amount of rrythrocyte prolidase. This caused a sharp reduction (58% ) in urinary peptide hydroxyproline but complete proteolysis was never achieved, thus providing additional evidence that the excretion of hydroxyproline peptides cannot be circumvented metabolically.” Exposure of ingested gelatin (denatured collagen) to the vigorous action of intestinal prolidase permitted a 7.V8 per cent excretion of hydroxyproline peptides into the urine of two women”” as compared to a 20 per cent excretion of hydroxyproline peptides after parenteral administration of gelatin to rats.” Although electron microscopic observations have implicated macrophages”’ and lysosomes”” in the process of tissue resorption, the lack of increase in urinary peptide hydroxyproline during the puerperium suggests that most of the uterine collagen is resorbed in a nondestructive manner. This conclusion is supported by isotopic evidence?:’ that another fibrous collagen (rat skin) can be depolymerized and reutilized.

ACKNOWLEDGMENTS We patient

thank with

for permitting us to study the Dr. R. Weisman, Department of Medicine, acute hemolysis. and Mrs. Corinne Pulphus for her fine technical assistance.

I’RINARY

PEPTIDE

3’ - .I

HYDROXYPROLINE

REFERENCES 1, Morrionc. T. G.. and Seifter, S.: Alteration in the collagen content of the human uteruh during pregnancy and post partum involution. J. Exp. Med. 115:?57. lY62. 2. Woessner, J. F.. and Brewer. T. H.: Formation and breakdown of collagen and elastin in the human uterus during pregnancy and post-partum involution. Biochem. J. H9:75, 1963. 3. Klein. L.: Urinary hydroxyproline during late pregnancy and post-partum involution. Metabolism 13:386. 1964. -1. Jelfrey. J. J.. and Gross. J.: Isolation and character-ization of a mammalian collagenolvtic enzyme. Fed. Proc. 26:670. 1967. 5 Weish. P. H.. and Klein, L.: The quantitative relationship of urinary peptide hydroxypruline excretion to collagen degradation. J. Clin. Invest. 48:l. 1969. 6. Fisher. R. B.: Protein Metabolism. London. Methuen. lY54, p. 13. 7. Klein. I... Albertsen. K., and Curtibs. P H.: Urinary hydroxyproline in hyperparathyroidiam: A study of three cases lath and without bone lesions. hletabolism 11:1023, 1962. X Meilman, E.. Urivetzky. M. M., and Rapoport. C. M.: Urinary hydroxyproline peptides. J. Clin. Invest. 42:40. 1963. Y. Slemons. J. M.: Metabolism during pregnancy. labor and the puerperium. Johns Hopkin\ Ho\p. Reports I?: I II. 1904. IO. Longridge, C. N.: Excretion of creatinin in lying-in women: with some remark\ on involution of the uterus. J. Obstet. Gynaec. Brit. Comm. 13:420. 1908. 1I. Montfort, 1.. and Perez-Tamayo, R.: Studies on uterine collagen during pregnancy and puerperium. Lab. Invest. IO: 1140. 1961. I?. Sjoel-dama. A.. Udenfriend. S.. Keiser. H. R.. and LeRoy, E. C.: Hydroxyproline and collagen metabolism: clinical implication\. Ann. Int. Med. 63:672. 1965.

13. Shetlar,

M. R.. Kelly,

K. H..

Foster.

I. V.. Shetlar, C. I... and Everett. M. I<. Serum polysaccharide levels in pregnanq. parturition and the postpartum state. Amel. J. Obstet. Gynec. 59: 1140. 1950. 14. Nir, I.. Sadovsky, E.. and Wei\cnE.: Serum bert. mucoprotein level\ in Israeli women during pregnancy. labor. and puerperium. Obstet. Gynec. 26:25X, 1965 15. Wvessner. J. I;.: Catabolism of cc~llagen and non-collagen protein in the ~;II uterus during postpartum involution. Kiothem. J. 83:304, 1962. 16. Gross. J.. and Lnpiere. C. M.: C (>Ilagenolytic activity in amphibian tissue\. 4 tissue culture assay. Proc. Nat. Acad. Sci. 48:1014, 1962. 17. Smith, E. L.. Davis. N. C‘.. Adam\. E., and Spackmnn, D. H.: The specifcity and mode of action of two metal-peptida\cs I/I McElroy, W. D., and Glass, B. (Ed\. I The Mechanism of Enzyme Action. Haltimore, Johns Hopkins Press. 1953. p. 2Yl.

18. Smith, F. I_.: The peptida\e\ 01 skeletal. heart. and uterine muscle. J. Blol. Chem. 173:553. 1948. 19. Christensen. H. N.. and Rafn. M. I.. Uptake of peptides hy a free-cell neopla\m. Cancer Res. 12:395. 1952. 20. Prockop. D. J.. Keiser. H. R.. .ind Sjoerdsma. A.: Castro-intestinal absor-ption and renal excretion of hydroxyproline peptide\. Lancet 2:527. 1962. 21. Parakkal. P. F.: Involvement ~)t’ mncrophnpes in collagen resorption. J. C‘zll Biol. 11 : 345. iY69. 22. Brandes. D.. osomes in uterine plasmic degradation collagen. J. Gerontol.

and Anton. E.: I y\involution: Intracytoof myofilament\ ;tnd 33:55. 1969.

23. Klein. L.: Reversible transformation of fibrous collagen to a soluble state iq vi\o. Proc. Nat. Acad. Sci. 62:920. 1969.