Urinary Proline Research Articles

Calibration of citrus intake assessed by food frequency questionnaires using urinary proline betaine in an observational study setting

BackgroundWhether observational study can be employed to establish calibration equations for self-reported dietary intake using food biomarkers is unknown. ObjectivesThis study aims to demonstrate the feasibility of obtaining calibration equations based on food biomarkers and 7-d diet records (7DDRs) to correct measurement errors of food frequency questionnaires (FFQs) in an observational study setting. MethodsThe study population consisted of 669 males and 749 females from the Women’s and Men’s Lifestyle Validation Studies. In the training set, the biomarker-predicted intake derived by regressing 7DDR-assessed intake on urinary proline betaine concentration was regressed on the FFQ-assessed intake to obtain the calibration equations. The regression coefficients were applied to the test set to calculate the calibrated FFQ intake. We examined total citrus as well as individual citrus fruits/beverages. ResultsUrinary proline betaine was moderately correlated with orange juice intake (Pearson correlation [r] = 0.53 for 7DDR and 0.48 for FFQ) but only weakly correlated with intakes of orange (r = 0.12 for 7DDR and 0.15 for FFQ) and grapefruit (r = 0.14 for 7DDR and 0.09 for FFQ). The FFQ-assessed citrus intake was systematically higher than the 7DDR-assessed intake, and after calibrations, the mean calibrated FFQ measurements were almost identical to 7DDR assessments. In the test set, the mean intake levels from 7DDRs, FFQs, and calibrated FFQs were 62.5, 75.3, and 63.2 g/d for total citrus; 41.6, 42.5, and 41.9 g/d for orange juice; 11.8, 24.3, and 12.3 g/d for oranges; and 8.3, 9.3, and 8.6 g/d for grapefruit, respectively. We observed larger differences between calibrated FFQ and 7DDR assessments at the extreme ends of intake, although, on average, good agreements were observed for all citrus except grapefruit. ConclusionsOur 2-step calibration approach has the potential to be adapted to correct systematic measurement error for other foods/nutrients with established food biomarkers in a cost effective way.

Assessing Repeated Urinary Proline Betaine Measures as a Biomarker of Usual Citrus Intake during Pregnancy: Sources of Within-Person Variation and Correlation with Reported Intake.

Proline betaine (Pro-B) has been identified as a biomarker of dietary citrus intake, yet gaps remain in its validation as a quantitative predictor of intake during various physiological states. This study quantified sources of within-individual variation (WIV) in urinary Pro-B concentration during pregnancy and assessed its correlation with the reported usual intake of citrus fruit and juice. Pro-B concentrations were determined by 1H-NMR spectroscopy in spot and 24-h urine specimens (n = 255) collected throughout pregnancy from women participating in the MARBLES cohort study. Adjusted linear or log mixed effects models quantified WIV and tested potential temporal predictors of continuous or elevated Pro-B concentration. Pearson or Spearman correlations assessed the relationship between averaged repeated biomarker measures and usual citrus intake reported by food frequency questionnaires. The proportion of variance in urinary Pro-B attributable to WIV ranged from 0.69 to 0.74 in unadjusted and adjusted models. Citrus season was a significant predictor of Pro-B in most analyses (e.g., adjusted β [95% CI]: 0.52 [0.16, 0.88] for non-normalized Pro-B), while gestational age predicted only non-normalized Pro-B (adjusted β [95% CI]: -0.093 [-0.18, -0.0038]). Moderate correlations (rs of 0.40 to 0.42) were found between reported usual citrus intake and averaged repeated biomarker measurements, which were stronger compared to using a single measurement. Given the high degree of WIV observed in urinary Pro-B, multiple samples per participant are likely needed to assess associations between citrus consumption and health outcomes.

Combining biomarker and food intake data: calibration equations for citrus intake

Measurement error associated with self-reported dietary intake is a well-documented issue. Combining biomarkers of food intake and dietary intake data is a high priority. The aim of this study was to develop calibration equations for food intake, illustrated with an application for citrus intake. Further, a simulation-based framework was developed to determine the portion of biomarker data needed for stable calibration equation estimation in large population studies. Calibration equations were developed using mean daily self-reported citrus intake (4-d semiweighed food diaries) and biomarker-derived intake (urinary proline betaine biomarker) data from participants (n=565) as part of a cross-sectional study. Different functional specifications and biomarker transformations were tested to derive the optimal calibration equation specifications. The simulation study was developed using linear regression for the calibration equations. Stability in the calibration equation estimations was investigated for varying portions of biomarker and intake data "qualities." With citrus intake, linear regression on nontransformed biomarker data resulted in the optimal calibration equation specifications and produced good-quality predicted intakes. The lowest mean squared error (14,354) corresponded to a linear regression model, defined with biomarker-derived estimates of intakes on the original scale. Using this model in a subpopulation without biomarker data resulted in an average mean±SD citrus intake of 81±66 g/d. The simulation study suggested that in large population studies, biomarker data on 20-30% of the subjects are required to guarantee stable estimation of calibration equations. This article is accompanied by a web application ("Bio-Intake"), which was developed to facilitate measurement error correction in self-reported mean daily citrus intake data. Calibration equations proved to be a useful instrument to correct measurement error in self-reported food intake data. The simulation study demonstrated that the use of food intake biomarkers may be feasible and beneficial in the context of large population studies.

Determinants of the urinary and serum metabolome in children from six European populations

BackgroundEnvironment and diet in early life can affect development and health throughout the life course. Metabolic phenotyping of urine and serum represents a complementary systems-wide approach to elucidate environment–health interactions. However, large-scale metabolome studies in children combining analyses of these biological fluids are lacking. Here, we sought to characterise the major determinants of the child metabolome and to define metabolite associations with age, sex, BMI and dietary habits in European children, by exploiting a unique biobank established as part of the Human Early-Life Exposome project (http://www.projecthelix.eu).MethodsMetabolic phenotypes of matched urine and serum samples from 1192 children (aged 6–11) recruited from birth cohorts in six European countries were measured using high-throughput 1H nuclear magnetic resonance (NMR) spectroscopy and a targeted LC-MS/MS metabolomic assay (Biocrates AbsoluteIDQ p180 kit).ResultsWe identified both urinary and serum creatinine to be positively associated with age. Metabolic associations to BMI z-score included a novel association with urinary 4-deoxyerythronic acid in addition to valine, serum carnitine, short-chain acylcarnitines (C3, C5), glutamate, BCAAs, lysophosphatidylcholines (lysoPC a C14:0, lysoPC a C16:1, lysoPC a C18:1, lysoPC a C18:2) and sphingolipids (SM C16:0, SM C16:1, SM C18:1). Dietary-metabolite associations included urinary creatine and serum phosphatidylcholines (4) with meat intake, serum phosphatidylcholines (12) with fish, urinary hippurate with vegetables, and urinary proline betaine and hippurate with fruit intake. Population-specific variance (age, sex, BMI, ethnicity, dietary and country of origin) was better captured in the serum than in the urine profile; these factors explained a median of 9.0% variance amongst serum metabolites versus a median of 5.1% amongst urinary metabolites. Metabolic pathway correlations were identified, and concentrations of corresponding metabolites were significantly correlated (r > 0.18) between urine and serum.ConclusionsWe have established a pan-European reference metabolome for urine and serum of healthy children and gathered critical resources not previously available for future investigations into the influence of the metabolome on child health. The six European cohort populations studied share common metabolic associations with age, sex, BMI z-score and main dietary habits. Furthermore, we have identified a novel metabolic association between threonine catabolism and BMI of children.

Diverse characteristics of the urinary excretion of amino acids in humans and the use of amino acid supplementation to reduce fatigue and sub-health in adults

BackgroundThe excretion of amino acids in urine represents an important avenue for the loss of key nutrients. Some amino acids such as glycine and histidine are lost in higher abundance than others. These two amino acids perform important physiological functions and are required for the synthesis of key proteins such as haemoglobin and collagen.MethodsStage 1 of this study involved healthy subjects (n = 151) who provided first of the morning urine samples and completed symptom questionnaires. Urine was analysed for amino acid composition by gas chromatography. Stage 2 involved a subset of the initial cohort (n = 37) who completed a 30 day trial of an amino acid supplement and subsequent symptom profile evaluation.ResultsAnalyses of urinary amino acid profiles revealed that three groups could be objectively defined from the 151 participants using k-means clustering. The amino acid profiles were significantly different between each of the clusters (Wilks’ Lambda = 0.13, p < 0.0001). Cluster 1 had the highest loss of amino acids with histidine being the most abundant component. Cluster 2 had glycine present as the most abundant urinary amino acid and cluster 3 had equivalent abundances of glycine and histidine. Strong associations were observed between urinary proline concentrations and fatigue/pain scores (r = .56 to .83) for females in cluster 1, with several other differential sets of associations observed for the other clusters.ConclusionsDifferent phenotypic subsets exist in the population based on amino acid excretion characteristics found in urine. Provision of the supplement resulted in significant improvements in reported fatigue and sleep for 81% of the trial cohort with all females reporting improvements in fatigue.Trial registrationThe study was registered on the 18th April 2011 with the Australian New Zealand Clinical Trials Registry (ACTRN12611000403932).

Abstract P280: Urinary Hippurate and Proline Betaine Excretion relative to Fleshy Fruit Intake and Blood Pressure: The INTERMAP Study

Background: Intakes of biologically complex fleshy fruits with substantial variations in nutritional composition may be reflected in urinary metabolite differences. We evaluated 24 hr urinary hippurate and proline betaine relative to individual fruit intake and blood pressure (BP) among free-living individuals in a population-based study. Methods: Cross-sectional epidemiological and metabonomic data from the International Study of Macro-/Micronutrients and Blood Pressure were used, available for 4,276 men and women ages 40-59 years from China, Japan, United Kingdom, and United States. Fleshy fruit intakes, according to Stern’s botanical classification, were calculated from four in-depth 24 hr dietary recalls. Urinary spectra from timed 24 hr urine collections were obtained by 1H nuclear magnetic resonance spectroscopy. Peak intensities of urinary hippurate (δ 7.84, doublet) and proline betaine (δ 3.11, singlet) were quantified. Mean urinary metabolite excretions across quartiles of energy-adjusted fruit intakes and corresponding P for trend were calculated. Country-specific multivariable linear regression coefficients between urinary metabolites and sum of metabolite-related fruits were estimated and pooled, weighted by inverse of their variance. Results: Age-sex-sample adjusted Pearson correlations (r) were: between hippurate and raw fruits 0.17, fruit juice 0.01, urinary potassium 0.19, and dietary fiber 0.15; between proline betaine and raw fruit 0.27, fruit juice 0.30, vitamin C 0.40, sugar 0.21, and dietary fiber 0.16; between hippurate and proline betaine 0.02. Significant multivariable adjusted P for trends (metabolome-wide significance level; P&lt;4x10-6) across quartiles of intake were found between total raw fruit, apple, kiwi fruit, nectarine, peach, plum, dried apricot, dried prune and hippurate; similarly between raw fruit, fruit juice, banana, grapefruit, kumquat, orange, satsuma, tangerine, grapefruit juice, orange juice and proline betaine. Correlations between sum of metabolite-related fruits and hippurate were 0.24 (0.10 for sum of remaining fruits) and 0.64 with proline betaine (0.03 for remaining fruits). After multivariable adjustment including BMI, differences in systolic BP were for urinary hippurate higher by 2SD -1.53 mm Hg (95% CI: -2.32,-0.75) and for sum of hippurate-related fruits -0.60 (-1.32,0.12). Urinary proline betaine and sum of proline betaine-related fruits were not associated with BP. Conclusion: Consumption of several individual fruits was related to urinary hippurate and proline betaine excretion. Higher urinary hippurate excretion was associated with lower systolic BP, but this was not found for hippurate-related fruits. This suggests that other polyphenol-rich foods and/or quantity may be important in this relationship.

DETERMINATION OF PROLINE, HYDROXYPROLINE, AND N–ETHYLGLYCINE IN URINE BY USING A NEW HPLC LABELING REAGENT, AND ITS APPLICATION IN DETECTION OF TUMOR MARKERS

According to previous reports, the concentration of urinary proline (Pro), hydroxyproline (Hyp), and N-ethylglycine (Neg) varies in association with various diseases, especially, Neg as a tumor marker was recently found in association with bone metastasis cancers. We developed a low-cost, highly sensitive precolumn high–performance liquid chromatography (HPLC) method for simultaneous determination of Pro, Hyp, and Neg in urine of patients with cancers, bone metastasis cancers, and bone metastasis cancers that have appeared after radiotherapy and chemotherapy treatment. The analytes in the urine were labeled with 4–methoxybenzenesulfonyl fluoride (MOBS–F) at 40°C. The derivatives were separated on a reversed-phase column by gradient elution and monitored with ultraviolet (UV) detection at 232 nm. The detection limits for Pro, Hyp, and Neg were 6.0 pmol/injection, 4.0 pmol/injection, and 50 pmol/injection (S/N = 3), respectively. To some extent, Neg levels in their urine show there were relevance to their health conditions or therapy progress. We believe that the developed method may be a promising measure for providing a useful reference to diagnose the illness and monitor the results for bone metastasis cancers after radiotherapy and chemotherapy treatment.

Simultaneous determination of proline and pipemidic acid in human urine by capillary electrophoresis with electrochemiluminescence detection.

Pipemidic acid is extensively used in the treatment of Gram-negative urinary tract infections, and the contents of proline in human urine vary in association with chronic uremia. The simultaneous determination of pipemidic acid and proline in human urine is of significance for quality control of the dosage and clinical study. The coupling of Ru(bpy)(3)(2+)-based electrochemiluminescence detection with capillary electrophoresis was developed for the simultaneous determination of proline and pipemidic acid in human urine. Parameters related to the separation and detection were investigated and optimized. The standard curves were linear between 0.1 and 90 µg mL(-1) for proline and between 0.4 and 100 µg mL(-1) for pipemidic acid. Underoptimized conditions, the detection limits (3σ) were 0.02 µg mL(-1) for proline and 0.06 µg mL(-1) for pipemidic acid. Relative standard derivations for the electrochemiluminescence intensity and the migration time were 3.2 and 0.9% for proline and 3.7 and 1.2% for pipemidic acid, respectively. The developed method was successfully applied to determine proline and pipemidic acid in human urine. The result showed that the content and decreasing rates of proline in urine for male were higher than that for female, and the content and decreasing rate of pipemidic acid in urine for male and female were consistent, respectively.

A rapid capillary electrophoresis with electrochemiluminescence method for the assay of human urinary proline and hydroxyproline

A novel simultaneous determination method for free and total proline (Pro) and hydroxyproline (Hyp) in human urine was developed, based on capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection, using tris-(2,2'-bipyridyl) ruthenium(II). Experimental conditions, such as the Ru(bpy)(3)2+ concentration, detection potentials, buffer concentration and pH in CE or in the ECL cell, injection voltage and time were investigated in detail. Under optimized conditions, the linear range, detection limit and sample recoveries for the method were 0.01-2 mmol/L (correlation coefficient, 0.9970), 4 micromol/L and 96.4-101.2% in human urine, respectively. The results show that the method has potential applications in monitoring the level of Pro and Hyp in body fluids from patients with bone disease, tumours or chronic uraemia.

Rapid screening of urinary proline–hydroxyproline dipeptide in bone turnover studies

In a recent report [J. Chromatogr. B 678 (1996) 165] a urinary hydroxyproline-containing peptide has been preliminarily suggested as a possible alternative to hydroxyproline (HP) determination in bone resorption studies. For this purpose a simple and practical procedure was developed for a rapid high-performance liquid chromatographic (HPLC) assay of the peptide in non-hydrolyzed urine samples. Hundreds of randomly selected urine samples were assayed for both the peptide and HP, the latter in hydrolyzed urine, and a high correlation between them was found. The promising results prompted us to search for the postulated biomarker of bone resorption in urine samples of postmenopausal women examined as osteoporosis suspects. As an alternative to the HPLC determination, an equally rapid procedure has been developed for the peptide assay using capillary gas chromatography (GC) with flame ionization detection (FID). By means of a solid-phase and a liquid–liquid phase extraction, involving ethyl chloroformate (ECF) as the derivatizing agent, two dipeptides and some urinary amino acids could be analyzed within 5 min. A high correlation between both HPLC and GC peptide assay was confirmed ( r=0.944) and the compound was identified as proline–hydroxyproline (PHP) dipeptide.

Selective and sensitive determination of urinary total proline and hydroxyproline by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of total urinary proline (Pro) and hydroxyproline (Hyp) by gas chromatography (GC) was developed. After acid hydrolysis of urine, primary amino compounds were eliminated by reaction with o-phthaldialdehyde. Subsequently, Pro and Hyp were converted into their N-dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB-5 capillary column. The derivatives were volatile and stable, giving single and symmetrical peaks. The detection limits for Pro and Hyp were 0.1 and 0.2 pmol injected, respectively. 3,4-Dehydroproline was used as an internal standard. The calibration curves for Pro and Hyp in the range 0.4–10 nmol were linear and sufficiently reproducible for quantitative determination. Overall recoveries of Pro and Hyp added to urine samples ranged from 93% to 103%. By using this method, Pro and Hyp in a small urine sample could be accurately and precisely determined without any influence from other constituent substances.

1589 VITAMIN D-DEFICIENT PROLINURIA IS EXPRESSED AT THE RENAL BRUSH BORDER MEMBRANE (BBM) INDEPENDENT OF PARATHYROID HORMONE(PTH)

Vitamin D deficiency is associated with a generalized amino-aciduria and phosphaturia. To study this tubulopathy weanling rats were fed several vitamin D deficient diets for 4-6 wks. 1) Very low Ca, 0.02% (VLC). 2) Low Ca, 0.47% (LC). 3) High Ca, 2.2%. and 4) Very Low P, 0.1% (VLP). Rats on VLC were given 500 pmoles 1,25(OH)2D3 for 2 days.Rats on VLC diet had 10 × elevated PTH and 2 X elevated urinary cAMP and 10 X prolinuria which did not change with 1,25(OH)2 D. Rats on LC and HC diets had attentuated changes 8 and 5 X in PTH and 1.7 and 1.8 X cAMP and 5 X and 11 X elevated prolinuria. Rats on VLP had normocalcemia (Ca=8.6±0.5) hypophosphatemia (PO4=6.4±0.6) normal PTH and urinary cAMP, but a 12 X elevated prolinuria, hypercaluria and no PO4 excretion.The Vmax & Km of proline uptake by BBMV on each diet was similar but a reduction in the peak of the overshoot at 30 sec. in all the vitamin D deficient diets was statistically lower than normal. 1,25(OH)2D3 corrects this defect in the VLC diet along with phosphaturia, and Ca excretion without affecting PTH or urinary cAMP. The peak overshoot correlated with the plasma PO4 (r=0.536, P< 0.02) but not Ca, cAMP, PTH or urinary proline. Thus, prolinuria of D-deficiency is expressed at the brush border membrane surface. H3-proline uptake by BBMV is independent of parathyroid status and appears to involve “leaky” membranes.

Urinary proline to hydroxyproline ratio varies with age

Urinary excretion of proline and hydroxyproline was studied in five groups of subjects, viz. controls, pituitary dwarfs, familial dwarfs, patients with fibrodysplasia ossificans progressive and patients with generalized scleroderma. The ratio Pro/Hyp in the urinary fraction precipitated with 5 parts of acetone (1 + 5 fraction) was close to one in children and youngsters in all the groups, while in adults it was higher than two. Total Pro/Hyp does not give an accurate index of age.

The relationship between the renal effects of cadmium and cadmium concentration in urine among the inhabitants of cadmium-polluted areas

The relationship between urinary cadmium concentration and the effects of cadmium on the kidney was studied in inhabitants of cadmium-polluted areas. As the indexes of the effects of cadmium on the kidney, total protein, retinol-binding protein, glucose, α-amino N, and proline in urine were employed. The prevalence of proteinuria, glucosuria, proteinuria with glucosuria, tubular proteinuria, and aminoaciduria increased distinctly with increasing cadmium in urine when the inhabitants were sorted according to their urinary cadmium concentrations expressed as micrograms per gram of creatinine. Probit analysis of urinary cadmium concentration (micrograms per gram of creatinine) and prevalence of renal effects showed linear regression lines. Therefore, cadmium concentration in urine expressed as micrograms per gram of creatinine seems to be useful in estimating the renal effects of cadmium exposure in the environment.

Proline and hydroxyproline excretion and vitamin C status in elderly human subjects.

1. Plasma and buffy-coat vitamin C, urinary proline, hydroxyproline, creatinine and total amino acid concentrations were meausred in 23 healthy elderly subjects at intervals of 3 months. 2. There was a strong positive correlation between plasma vitamin C and buffy-coat vititamin C. 3. There were not significant correlations between plasma or buffy-coat vitamin CPAMIN C. 3. There were not significant correlations between plasma or buffy-coat vitamin C and total urinary hydroxyproline, whether expressed on a creatinine basis or on a total amino acid basis. Similarly, no significant correlations could be detected involving the proline/hydroxyproline ratio in urine hydrolysates. 4. There was a significant negative correlation between plasma or buffy-coat vitamin C and total urinary proline, when expressed per unit of total urinary proline, when expressed per unit of total urinary proline, when expressed per unit of total amino acids in the hydrolysates. This correlation was not observed with unhydrolysed urine, and it appeared to reside in the diffusible fraction, part of whose proline could be liberated by prolidase digestion. In addition, in the man, there was some evidence for a positive correlation between plasma or buffy-coat vitamin C and the ratio of total urinary amino acids to creatinine. 5. These results support the view that poor vitamin C status in elderly humans may be associated with a defect in collagen proline hydroxylation, reflected by increased excretion of proline-rich, collagen-derived peptides. If this interpretation is correct, it indicates a potential defect in connective tissue repair, and could form the basis of a functional test for subclinical vitamin C deficiency.

THE BONE HISTOLOGY IN THE SYNDROME OF MEDULLARY STENOSIS OF TUBULAR BONES, DWARFISM AND HYPOCALCEMIA

The pathogenesis of this syndrome has not been elucidated. The present case-a 12 years old boy-demonstrated dwarfism, stenosis of medullary spaces of bones, intracranial calcifications and permanent hypocalcemic hyperphosphatemic tetany. Thyroxine and growth hormone levels were normal. Plasma parathormone was elevated. The response to parathormone was normal: a significant rise in serum Ca, a fall in serum phosphate, an increase is renal phosphate clearance, and an increase in urinary proline and OH-proline. Microradiographs of cross sections from the fibula shaft showed normal bone structure and density. However there was remarkably little remodeling activity: only one or two forming osteones in the entire section and only one resorption cavity. This is the first child with this syndrome in whom the bone is examined.

THE BONE HISTOLOGY IN THE SYNDROME OF MEDULLARY STENOSIS OF TUBULAR BONES, DWARFISM AND HYPOCALCEMIA

The pathogenesis of this syndrome has not been elucidated. The present case-a 12 years old boy-demonstrated dwarfism, stenosis of medullary spaces of bones, intracranial calcifications and permanent hypocalcemic hyperphosphatemic tetany. Thyroxine and growth hormone levels were normal. Plasma parathormone was elevated. The response to parathormone was normal: a significant rise in serum Ca, a fall in serum phosphate, an increase is renal phosphate clearance, and an increase in urinary proline and OH-proline. Microradiographs of cross sections from the fibula shaft showed normal bone structure and density. However there was remarkably little remodeling activity: only one or two forming osteones in the entire section and only one resorption cavity. This is the first child with this syndrome in whom the bone is examined.

Urinary free proline and collagen metabolites in newborns, healthy children, and in collagen diseases

The urinary output of free and peptide-bound imino acids was studied in 41 newborns and 48 children aged 3–14 years; 20 of these latter suffered from various collagen diseases. Quantitative data about free urinary proline are given. The relationship between free and total urinary proline as well as hydroxyproline and plasma proline was studied. The urinary output of total and free imino acids was increased from the 1st through the 15th day of life. There was a significant decrease in the urinary output of both free and total imino acids in children with collagen disease, though plasma proline levels were normal.

Intravenous proline tolerance in osteogenesis imperfecta.

The biological half-life of proline in blood has been determined in osteogenesis imperfecta (188±30 min., mean ±1 SD), in pediatric controls (409±130 min.), and in adult control subjects (511±163 minutes). Renal clearances (ml./min./1.73 m 2 ) of free and bound proline have been calculated for the 3 groups. The loss of free proline in urine does not appear to be sufficient to account for the short half-life in blood. Creatinine clearances (corrected to 1.73 m 2 ) were not significantly different in the 3 groups studied. The difference in proline half-life in blood in patients with osteogenesis imperfecta and in normal subjects can most probably be explained by an increased rate of proline uptake in cells or by more rapid incorporation of the amino acid into intermediary metabolic pathways in patients with the disorder. The higher level of bound proline in urine in patients with the disorder may be a reflection of increased turnover or catabolism of collagen.