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We characterized the degradation products of recently synthesized collagen present in skins of control and diabetic rats. Specifically, the TCA-soluble fractions of homogenized skins from control and diabetic rats (killed 1 and 4 hours after [3H]-proline injection) were fractionated by molecular sieve chromatography, and eluted fractions were analyzed for hydroxyproline and [3H]-hydroxyproline. Free [3H]-hydroxyproline was the principal (greater than 95%), low molecular weight (greater than 2000 daltons), [3H]-hydroxyproline-containing material eluted from the molecular sieve column, this amount representing approximately 80% (controls) and approximately 87% (diabetics) of [3H]-hydroxyproline-containing material in TCA-soluble fractions of skin homogenates. These observations are similar to those from the intracellular degradation of cellular and secretory proteins in that the principal--almost exclusive--degradation product was the free amino acid. The free hydroxyproline had a greater specific radioactivity than that in any other [3H]-hydroxyproline-containing fraction (soluble and insoluble, see below); furthermore, the total radioactivity of free [3H]-hydroxyproline was greater at 1 hour than 3 hours later. These two properties (identity with free amino acid; time-dependent decrease in amounts) are consistent with [3H]-hydroxyproline arising from the intracellular degradation of procollagen. The [3H]-hydroxyproline-containing material eluting before free hydroxyproline (designated peptidyl [3H]-hydroxyproline) was similar to free [3H]-hydroxyproline in terms of specific radioactivity and the time-dependent decreases of specific and total radioactivities, these similarities indicating that the peptidyl [3H]-hydroxyproline are intermediates in the degradative pathway of procollagen to free amino acids. Results for control and diabetic rats were qualitatively similar, with regard to the inter-fraction ratios of specific radioactivities and their time-dependent changes. However, the degradative process, as assessed by the release of free and peptidyl [3H]-hydroxyproline, was dramatically enhanced by the diabetic state, extending our previous results based on analyses of uncharacterized degradation products.
Changes in prostatic collagen were measured in Sprague-Dawley rats to gain further insight into the relationship between this stromal component and androgen mediated prostatic growth. Regulation of prostatic collagen by other endocrine factors was also studied. Collagen content per prostate was estimated by determination of tissue levels of hydroxyproline. The 1st experiment examined changes in the content of hydroxyproline in the prostate during pre- and post-pubertal growth with the use of rats between 21 and 80 days of age. As the animals grew, their prostatic weights and hydroxyproline contents increased in a parallel fashion (correlation coefficient R = 0.977, p less than 0.01). In the 2nd experiment, rats were castrated for a period up to 28 days. The hydroxyproline content in the prostate did not change significantly by castration despite a marked decrease in prostatic weights. Results of the 3rd experiment indicated that castration-hypophysectomy or castration-hypophysectomy plus estrogen treatment did not significantly change the content of prostatic hydroxyproline from that in the untreated intact animals. The 4th experiment studied the effect of the collagen synthesis inhibitor, cis-4-hydroxyproline, on prostatic growth. Subcutaneous injection of cis-4-hydroxyproline to castrated testosterone treated rats caused a significantly slower increase in total ventral prostatic weights and contents of protein, DNA and hydroxyproline than those of saline treated controls. This inhibition in prostatic growth is unlikely to be related to any antiandrogenic effect of cis hydroxyproline as the protein/DNA ratio in the prostate was the same for both saline and cis-4-hydroxyproline treated groups. Electron microscopic studies revealed that cis-4-hydroxyproline treatment resulted in a derangement of the basement membrane in the ventral prostate. The above results suggest that collagen plays an important role in limiting prostatic growth since inhibition of collagen synthesis by cis-4-hydroxyproline retarded the testosterone induced increase in prostatic weight, DNA, protein and hydroxyproline content in the prostate of rats.
Hydroxyproline-containing polypeptides were isolated from urine by filtration on polyacrylamide gel Bio-Gel P-2. This fraction was quantitatively and qualitatively similar to the nondialysable hydroxyproline in urines of normal adult subjects, patients with chronic uremia, and with primary or secondary hyperparathyroidism. Hydroxyproline polypeptides isolated from urines of patients with extensive Paget's disease or from normal growing subjects contained a group of smaller polypeptides slightly retarded on Bio-Gel P-2 which were not retained by the dialysis membrane Spectrapor-TM. In these two groups of subjects the values of hydroxyproline polypeptides were 35-45 percent higher than those of dialysable hydroxyproline. Increased excretion of hydroxyproline polypeptides was found in chronic uremia, primary and secondary hyperparathyroidism, Paget's bone disease and in normal growing subjects. With the notable exception of chronic uremia the excretion of hydroxyproline polypeptides paralleled that of total and oligopeptidic hydroxyproline. In some cases of chronic uremia the polypeptidic hydroxyproline represented the predominant fraction. On long columns of Bio-Gel P-6 hydroxyproline polypeptides separated into several peaks. The chromatographic pattern was rather characteristic for chronic uremia, secondary hyperparathyroidism, Paget's bone disease and physiological growth. Ahydroxyproline fraction of similar chromatographic properties was isolated from blood serum after deproteinisation with heat or ethanol at pH 4.8. High serum levels of this hydroxyproline fraction were observed in pathological conditions with increased urinary excretion of polypeptidic and nondialysable hydroxyproline.
Values for total 3-hydroxyproline and 4-hydroxyproline were obtained from 24-h urine specimens of 18 healthy human subjects of both sexes, whose ages ranged from the first to the sixth decade in age. Urinary 3-hydroxyproline levels, not earlier described to our knowledge, were determined by an isotope-dilution method requiring considerable purification and utilizing the amino acid analyzer for final measurement. 3-Hydroxyproline averaged 3% of the corresponding 4-hydroxyproline in individual urine samples. Like 4-hydroxyproline, 3-hydroxyproline excretion is increased in the second decade, and there is generally good correlation between the two values in individual urines. A hydroxyprolinemic subject excreting greatly elevated 4-hydroxyproline levels did not excrete excessive 3-hydroxyproline, consistent with independent catabolic pathways for the two compounds. 3-Hydroxyproline appears to be selectively excreted relative to 4-hydroxyproline when compared with the probable total body content of each amino acid. Possible explanations are: a more rapid turnover of basement membrane collagen than interstitial collagen or, alternatively, relatively greater resistance to the proteolytic cleavage of peptides containing 3-hydroxyproline.
A method for determining the endogenous urinary excretion levels of both 3-hydroxyproline and 4-hydroxyproline that may be useful for cancer screening of the general population and at the workplace is evaluated in this report. The excretion levels of 3-hydroxyproline and 4-hydroxproline were estimated in 97 patients with cancer and in 99 patients with various nonmalignant diseases and were compared with those of 211 healthy persons. Measurable 3-hydroxyproline peaks (by amino acid autoanalyzer) were absent from 93 samples from 211 healthy persons (44%), 50 of 99 patients with nonmalignant disease (50%), and 10 of 96 patients with cancer (10%). The levels of both 3-hydroxyproline and 4-hydroxyproline in cancer patients were significantly higher than those in healthy persons (p < 0.001 and p < 0.01, respectively) and those in patients with nonmalignant diseases (p < 0.05 and p < 0.01, respectively). Cancer patients were classified into three groups according to grade of cancer growth and invasion. The sensitivity of 3-hydroxyproline was 44% and higher than that of 4-hydroxyproline for the detection of stage II cancers (no distant metastasis); the sensitivities of both hydroxyprolines for the detection of stage I (very early cancer) were low. The specificity of these assays for healthy persons and patients with nonmalignant disease was 96% and 92% for 3-hydroxyproline, and 97% and 79% for 4-hydroxyproline, respectively. Urinary 3-hydroxyproline level should be further investigated as a cancer screening method for healthy persons in the community or the workplace, but appears unlikely to detect many cancers in the earliest stages.
The excretion of peptide-bound hydroxyproline before and after gelatin loading was evaluated in two children with prolidase deficiency, two adult heterozygotes, and normal controls. On a low hydroxyproline 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 (82.8% 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 hydroxyproline.
Synovial fluid and serum hydroxyproline fractions were investigated in patients with osteoarthritis and microcrystalline arthritis. Synovial fluid dialysable hydroxyproline levels are higher than serum levels in both conditions. Synovial fluid total and dialysable hydroxyproline are higher in microcrystalline arthritis than in osteoarthritis, while non-dialysable hydroxyproline values are similar in both conditions. In microcrystalline arthritis, synovial fluid dialysable hydroxyproline and polymorphonuclear leukocyte counts closely parallel each other. Irrespective of the type of arthropathy, synovial fluid dialysable hydroxyproline levels correlate with urinary hydroxyproline excretion. While the data suggest overproduction of dialysable hydroxyproline by joints in both conditions, the overproduction appears to be mediated by polymorphonuclear leukocytes in microcrystalline arthritis only. The ratio of serum to synovial total hydroxyproline are further suggestive of a possible differentiation between osteoarthritis and microcrystalline arthritis. In the conditions governing the present study, urinary hydroxyproline may be used as an index of joint tissue collagen resorption. Finally the significance of synovial fluid and serum non-dialysable hydroxyproline is discussed.
To clarify the significance of free and small peptide-bound hydroxyproline synthesis in ethanol-induced liver injury, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol-soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. The synthesis of free and small peptide-bound [14C]hydroxyproline was 11.1 +/- 2.0 dpm x 10(-4)/g liver/3 hr and the synthesis of protein-bound [14C]hydroxyproline was 10.1 +/- 3.3 dpm x 10(-4)/g liver/3 hr in control rat liver. In the ethanol-fed rat liver, the synthesis of free and small peptide-bound [14C]hydroxyproline significantly increased 1.5-fold and the synthesis of protein-bound [14C]hydroxyproline significantly increased 1.6-fold, while the hepatic collagen content did not change. There was a significant correlation between free and small peptide-bound [14C]hydroxyproline synthesis and protein-bound [14C]hydroxyproline synthesis. These results suggest that free and small peptide-bound hydroxyproline synthesis plays an important role in regulating the content of hepatic collagens.
To clarify the process of free and small peptide-bound hydroxyproline synthesis in hepatic fibrogenesis, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. In control rat liver, the amount of free and small peptide-bound [14C]hydroxyproline synthesized was 13.1 +/- 2.6 10(-4) x dpm/g liver/3 hr. In the CCl4-treated rat liver, where the hepatic hydroxyproline content was increased 4.6-fold, the protein-bound [14C]hydroxyproline synthesis was significantly increased 1.5-fold, but free and small peptide-bound [14C]hydroxyproline synthesis was decreased into 70%. There was a significant inverse correlation between free and small peptide-bound [14C]hydroxyproline synthesis, and hepatic hydroxyproline content. These results suggest that the combination of an increase in collagen synthesis and a decrease in free and small peptide-bound [14C]hydroxyproline synthesis contributes to rapid accumulation of collagen in hepatic fibrosis.
It is possible to assess bone resorption from a determination of urinary excretion of hydroxyproline, which is the specific amino-acid of collagen. As dietary collagen affects 24-hour urinary excretion of hydroxyproline, it has been stated that the urine should be collected under a gelatin-free diet. A new sampling method was described in the present paper for the determination of hydroxyproline in urine, which could eliminate the affection of dietary collagen by simple fasting. The method was useful for the evaluation of bone metabolism in patients with parathyroid disorders. 10 patients with primary hyperparathyroidism (3 skeletal types and 7 non-skeletal types), 3 patients with idiopathic hypoparathyroidism and 5 normal subjects were studied. It was found that the urinary excretion of hydroxyproline increased at night and diminished during the day in patients with primary hyperparathyroidism as well as in normal subjects, but this diurnal rhythm was not clear in a patient with idiopathic hypoparathyroidism. A pilot study revealed that 10 g gelatin administered orally did not affect the urinary excretion of hydroxyproline after a 12-hour fast. Therefore, 2-hour urine samples (700 h-900 h) were collected, and blood samples were drawn at 800 h after a 13-hour fasting from 1800 h on the previous day to 700 h in the morning studied. The urinary excretion of hydroxyproline was expressed as follows: HOP (microgram/ml)/Cr(mg/dl). The 2-hour urinary excretion of hydroxyproline thus determined was highly correlated with that determined in 24-hour urine collected under a gelatin-free diet (r = 0.995, p less than 0.001) and with the total serum alkaline phosphatase activity (r = 0.987, p less than 0.001). The levels of 2-hour urinary excretion of hydroxyproline in normal subjects were 0.18-0.28 in range, and those in patients with the skeletal type of primary hyperparathyroidism were high. However, the levels were not always higher than those in the patients with the non-skeletal type, in which cases the 2-hour excretion of hydroxyproline was higher than 0.50 except in one patient. The 9 patients with primary hyperparathyroidism who had elevated levels of the 2-hour urinary excretion of hydroxyproline showed tetany after parathyroidectomy.(ABSTRACT TRUNCATED AT 400 WORDS)
A procedure has been developed which can detect the hydroxyproline isomers trans-4-hydroxyproline (Hyp), trans-3-hydroxyproline, cis-4-hydroxyproline, and cis-3-hydroxyproline present in hydrolysates of collagens. The method involves hydrolyzing collagen, and reacting the primary amino acids with o-phthaladehyde (OPA) and the hydroxyprolines and proline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) which combines specifically with secondary amino acids. The proline and hydroxyprolines are then separated by thin-layer chromatography and quantified by using a scanning spectrofluorometer. The method was used to show that both trans-4-L-hydroxyproline and trans-3-L-hydroxyproline were epimerized as a function of hydrolysis time to the cis isomers. An appreciable amount of trans-3-Hyp was degraded. Hydrolysis with 6 N HCl in the presence of 6% trichloroacetic acid gave greater epimerization than the 6 N HCl alone. Alkaline hydrolysis in 0.2 M Ba(OH)2 caused more epimerization of trans-4-Hyp and trans-3-Hyp compared with acid hydrolysis but less degradation, so that alkaline hydrolysis is proposed for the evaluation of trans-3-Hyp, provided that the total of the cis and trans isomers be considered in this case.
A study has been made of the hydroxyproline-induced inhibition of elongation of Avena coleoptile tissues. The isomers of 4-hydroxyproline differ in their effectiveness; only the L isomers are growth inhibitors with the cis form (allohydroxyproline) being more effective than the trans form (hydroxyproline). Hydroxyproline differs from other amino acid antagonists and protein synthesis inhibitors in respect to 2 characteristics of the growth inhibition. First, a certain increment of auxin-induced elongation must take place following addition of hydroxyproline before the growth is inhibited. In contrast, pretreatment with other amino acid antagonists or protein synthesis inhibitors completely eliminates the ability of Avena coleoptile sections to respond to auxin. Secondly, sucrose markedly increases the magnitude of the hydroxyproline inhibition; i.e., sucrose acts to inhibit rather than promote growth when in the presence of hydroxyproline. It appears that hydroxyproline is a specific inhibitor for the synthesis of some factor which is utilized in elongation. Following addition of hydroxyproline, auxin-induced elongation continues until the pool of this factor is exhausted; then elongation is inhibited.
1. Urinary dialysable and non-dialysable hydroxyproline, which are considered good indices of bone resorption and neoformation respectively, were determined in rats under conditions that modify skeleton metabolism, such as body growth and parathyroid or calcitonin administration. It was also investigated whether dialysable and non-dialysable hydroxyproline excretions showed significant circadian fluctuations in rats of different ages.2. Dialysable hydroxyproline excretion sharply decreased from the first to the fifth months of age and underwent further gradual reduction up to the fourteenth month of life. Non-dialysable hydroxyproline excretion followed a smoother decrease up to the fifth month, then remained constant. Urinary excretion of non-dialysable hydroxyproline expressed as a percentage of the total hydroxyprolinuria (n.d.%) slowly increased with advancing rat age.3. In 2-, 4- and 6-month old rats, dialysable hydroxyproline excretion showed significant circadian fluctuations with minima and maxima at the end of the dark and light fraction of the cycle respectively. Daily fluctuations were greater in young and adult rats (50-65% of the respective average levels) than in 4-month old rats (25%). Non-dialysable hydroxyproline excretion followed similar but less pronounced patterns. Significant circadian fluctuations of n.d.% were detectable only in 2- and 4-month old rats, with peaks at 04.00-05.00 hr, thus indicating that the bone formation/resorption ratio increased in the nocturnal fraction of the cycle.4. Young rats administered with calcitonin exhibited reduced levels of urinary dialysable but not of non-dialysable hydroxyproline when the hormone was given at 13.30 hr. No changes were observed when calcitonin was injected at 19.30 hr. On the contrary, both diurnal and nocturnal parathyroid hormone administration to young rats caused increased levels of dialysable and non-dialysable hydroxyproline of the same magnitude.
Urinary glycosaminoglycan and hydroxyproline excretion was studied in 11 patients with clear evidence of Paget's disease of bone. Urinary hydroxyproline, cetyl pyridinium chloride (CPC)-precipitable uronic acid and CPC-precipitable hexosamine were expressed as ratios to urinary creatinine. Urine samples were concentrated x 1000 by vacuum dialysis and the glycosaminoglycans examined by electrophoresis on cellulose acetate followed by staining with alcian blue. All the cases studied showed markedly raised hydroxyproline excretion, whereas the uronic acid excretion was normal or only slightly raised in 10 of the 11 cases studied. One patient who had a raised uronic acid and raised hydroxyproline concentration was shown to have osteosarcoma as a complication of Paget's disease. THE VERY HIGH HYDROXYPROLINE: creatinine ratio in all cases of Paget's disease (mean 241.8 mmol hydroxyproline/mol creatinine) contrasted sharply with the cases of disseminated neoplasm, where the ratio was either normal or slightly raised (mean 29.3 mmol hydroxyproline/mol creatinine). The ratio of hydroxyproline to CPC-precipitable uronic acid was also markedly raised in cases of Paget's disease (mean 77.3 mmol hydroxyproline/mmol uronic acid) and was lower in the neoplastic group (mean 14.1 mmol hydroxyproline/mmol uronic acid) but showed no advantage over the hydroxyproline: creatinine ratio in differentiating the two groups. THE URINARY HYDROXYPROLINE: creatinine ratio promises to be of value in differentiating between Paget's disease of bone and neoplastic invasion of bone. A marked rise in CPC-precipitable uronic acid excretion alone is more suggestive of neoplastic invasion of bone, and if associated with a marked increase in hydroxyproline excretion, it raises the possibility of neoplastic change in Paget's disease of bone. The results of this study also suggest that bone collagen, rather than bone tissue in general, is primarily affected in Paget's disease.
The excretion of urinary hydroxyproline has been measured before mastectomy in 342 patients presenting with breast cancer for the first time to Guy's Hospital. The first 106 women were maintained on a gelatine-free diet whilst the remainder were on unrestricted diet. In both dietary groups hydroxyproline levels or the ratio of hydroxyproline to urinary creatinine were not related to pathological stage or histological grade. The time between initial presentation and subsequent bone metastases was negatively and significantly associated with hydroxyproline excretion (P less than 0.05) and the ratio of hydroxyproline to creatinine (P less than 0.01) in women on a gelatine-free diet. A similar, but not significant, trend was observed in patients on unrestricted diet. Although hydroxyproline excretion was related to the time to onset of bone metastases the amount of hydroxyproline excreted by these patients was not significantly different from patients who had recurrences at sites other than bone or patients who were disease-free up to 5 years after initial diagnosis. The conclusion is that hydroxyproline is of little value in the early detection of bone metastases.
We analysed over a period of 30-32 days the daily total hydroxyproline and creatinine excretine in urine from 9 healthy, normally fed 4 to 6 year-old children (2 girls, 7 boys). The average urinary hydroxyproline excretion was 45.6 mg/24 hr, with a coefficient of variation of 25.6%. Urinary hydroxyproline for individual children showed distinct differences from day to day, which were independent of urine volume. There were significant differences between the mean values for urine hydroxyproline in individual children, which were independent of age. The average creatinine in urine was 346/24 hr with a coefficient of variation of 17.7%. The hydroxyproline-index did not define the nutritional state of these normally developed children on a normal diet. Dietary hydroxyproline contributed 7.4% to the total urinary hydroxyproline in our investigation. There was a close correlation between urine hydroxyproline excretion and growth velocity in each child.
The effect of dietary control on the day-to-day variation in total hydroxyproline excretion has been examined in two studies. In the first, a normal volunteer ate a controlled diet containing varying gelatin supplements for several weeks. In the second, the effect of removing hydroxyproline-containing foods from the diets of 8 volunteers was examined. Both studies confirm that the day-to-day variation in total hydroxyproline excretion falls when the gelatin content of the diet is decreased, whether the results are expressed as total hydroxyproline excretion rate or as the total hydroxyproline: creatinine ratio. This fall in variation takes place within 24 h of dietary control beginning and therefore longer periods of dietary restrictions to achieve optimum precision in the measurement of total hydroxyproline are unnecessary. For some analytical methods, results are better expressed as the ratio total hydroxyproline:creatinine than as the total hydroxyproline excretion rate.