Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Abstract

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research. Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research. Glycosaminoglycans (GAGs) are a family of long, linear polysaccharides consisting of repeating disaccharide units (1Chen Y.-H. Narimatsu Y. Clausen T.M. Gomes C. Karlsson R. Steentoft C. Spliid C.B. Gustavsson T. Salanti A. Persson A. Malmström A. Willén D. Ellervik U. Bennett E.P. Mao Y. et al.The GAGOme: A cell-based library of displayed glycosaminoglycans.Nat. Methods. 2018; 15: 881-888Google Scholar). Different classes of GAGs have been characterized. In humans, the most prevalent classes are chondroitin sulfate (CS) [(→3)-β-D-GalNAc(1→4)-β-D-GlcA or α-L- IdoA(1→], heparan sulfate (HS) [(→4)-α-D-GlcNAc or α-D- GlcNS(1→4)-β-D-GlcA or α-L-IdoA (1→], and hyaluronic acid (HA) [(→3)-β-D-GlcNAc(1 → 4)-β-D-GlcA(1→] where GalNAc is N-acetylgalactosamine, GlcA is glucuronic acid, IdoA is iduronic acid, GlcNAc is N-acetylglucosamine, and GlcNS is N-sulfoglucosamine. Glucuronic acid can be further modified by sulfation in up to three sites. Chondroitin Sulfate and HS disaccharides can each be further modified with O-sulfo groups, typically in up to three positions in humans. The resulting sulfation motifs confer GAGs highly diverse biological functions that are essential for healthy human development and physiology (2Soares da Costa D. Reis R.L. Pashkuleva I. Sulfation of glycosaminoglycans and its implications in human health and disorders.Annu. Rev. Biomed. Eng. 2017; 19: 1-26Google Scholar). The panel of GAG motifs resulting from the diversity in structure and concentration of GAGs is collectively referred to as GAGome. Alterations in the physiological function of GAGs have been associated with several diseases ranging from mucopolysaccharidosis, a group of rare metabolic disorders caused by genetic defects in lysosomal enzymes that degrade GAGs, to complex diseases such as sepsis, rheumatoid arthritis, and cancer (3Khan S.A. Mason R.W. Giugliani R. Orii K. Fukao T. Suzuki Y. Yamaguchi S. Kobayashi H. Orii T. Tomatsu S. Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis.Mol. Genet. Metab. 2018; 125: 44-52Google Scholar, 4Schmidt E.P. Overdier K.H. Sun X. Lin L. Liu X. Yang Y. Ammons L.A. Hiller T.D. Suflita M.A. Yu Y. Chen Y. Zhang F. Cothren Burlew C. Edelstein C.L. Douglas I.S. et al.Urinary glycosaminoglycans predict outcomes in septic shock and acute respiratory distress syndrome.Am. J. Respir. Crit. Care Med. 2016; 194: 439-449Google Scholar, 5Wang J.Y. Roehrl M.H. Glycosaminoglycans are a potential cause of rheumatoid arthritis.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14362-14367Google Scholar, 6Gatto F. Volpi N. Nilsson H. Nookaew I. Maruzzo M. Roma A. Johansson M.E. Stierner U. Lundstam S. Basso U. Nielsen J. Glycosaminoglycan profiling in patients’ plasma and urine predicts the occurrence of metastatic clear cell renal cell carcinoma.Cell Rep. 2016; 15: 1822-1836Google Scholar). Plasma and urine GAGomes have been proposed as promising biomarkers for early noninvasive diagnostics (6Gatto F. Volpi N. Nilsson H. Nookaew I. Maruzzo M. Roma A. Johansson M.E. Stierner U. Lundstam S. Basso U. Nielsen J. Glycosaminoglycan profiling in patients’ plasma and urine predicts the occurrence of metastatic clear cell renal cell carcinoma.Cell Rep. 2016; 15: 1822-1836Google Scholar). Despite the potential role of GAGs for clinical applications, the measurements of the GAGome has been limited to very small sample sizes (ranging from 3 healthy donors in (7Wei W. Niñonuevo M.R. Sharma A. Danan-Leon L.M. Leary J.A. A comprehensive compositional analysis of heparin/heparan sulfate-derived disaccharides from human serum.Anal. Chem. 2011; 83: 3703-3708Google Scholar) to 25 in (6Gatto F. Volpi N. Nilsson H. Nookaew I. Maruzzo M. Roma A. Johansson M.E. Stierner U. Lundstam S. Basso U. Nielsen J. Glycosaminoglycan profiling in patients’ plasma and urine predicts the occurrence of metastatic clear cell renal cell carcinoma.Cell Rep. 2016; 15: 1822-1836Google Scholar)), in predominantly retrospective and selected donors, with different analytical techniques performed within academic laboratories (3Khan S.A. Mason R.W. Giugliani R. Orii K. Fukao T. Suzuki Y. Yamaguchi S. Kobayashi H. Orii T. Tomatsu S. Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis.Mol. Genet. Metab. 2018; 125: 44-52Google Scholar, 4Schmidt E.P. Overdier K.H. Sun X. Lin L. Liu X. Yang Y. Ammons L.A. Hiller T.D. Suflita M.A. Yu Y. Chen Y. Zhang F. Cothren Burlew C. Edelstein C.L. Douglas I.S. et al.Urinary glycosaminoglycans predict outcomes in septic shock and acute respiratory distress syndrome.Am. J. Respir. Crit. Care Med. 2016; 194: 439-449Google Scholar, 6Gatto F. Volpi N. Nilsson H. Nookaew I. Maruzzo M. Roma A. Johansson M.E. Stierner U. Lundstam S. Basso U. Nielsen J. Glycosaminoglycan profiling in patients’ plasma and urine predicts the occurrence of metastatic clear cell renal cell carcinoma.Cell Rep. 2016; 15: 1822-1836Google Scholar, 7Wei W. Niñonuevo M.R. Sharma A. Danan-Leon L.M. Leary J.A. A comprehensive compositional analysis of heparin/heparan sulfate-derived disaccharides from human serum.Anal. Chem. 2011; 83: 3703-3708Google Scholar, 8Gatto F. Blum K.A. Hosseini S.S. Ghanaat M. Kashan M. Maccari F. Galeotti F. Hsieh J.J. Volpi N. Hakimi A.A. Nielsen J. Plasma glycosaminoglycans as diagnostic and prognostic biomarkers in surgically treated renal cell carcinoma.Eur. Urol. Oncol. 2018; 1: 364-377Google Scholar, 9Gatto F. Maruzzo M. Magro C. Basso U. Nielsen J. Prognostic value of plasma and urine glycosaminoglycan scores in clear cell renal cell carcinoma.Front. Oncol. 2016; 6: 253Google Scholar). These limitations can be attributed to the historical lack of effective analytical methods until recently (10Wei W. Miller R.L. Leary J.A. Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: Evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes.Anal. Chem. 2013; 85: 5917-5923Google Scholar, 11Volpi N. Galeotti F. Yang B. Linhardt R.J. Analysis of glycosaminoglycan-derived, precolumn, 2-aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection.Nat. Protoc. 2014; 9: 541-558Google Scholar, 12Han X. Sanderson P. Nesheiwat S. Lin L. Yu Y. Zhang F. Amster I.J. Linhardt R.J. Structural analysis of urinary glycosaminoglycans from healthy human subjects.Glycobiology. 2020; 30: 143-151Google Scholar, 13Sun X. Li L. Overdier K.H. Ammons L.A. Douglas I.S. Burlew C.C. Zhang F. Schmidt E.P. Chi L. Linhardt R.J. Analysis of total human urinary glycosaminoglycan disaccharides by liquid chromatography–tandem mass spectrometry.Anal. Chem. 2015; 87: 6220-6227Google Scholar, 14Tomatsu S. Shimada T. Mason R.W. Kelly J. LaMarr W.A. Yasuda E. Shibata Y. Futatsumori H. Montaño A.M. Yamaguchi S. Suzuki Y. Orii T. Assay for glycosaminoglycans by tandem mass spectrometry and its applications.J. Anal. Bioanal. Tech. 2014; 2014: 006Google Scholar), which proved hard to standardize and expensive to run. As a result, the GAGome measurements reported in the literature for healthy subjects are widely variable and cannot be consistently used as reference for physiology and biomarker research. In this study, we took advantage of a standardized analytical method using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS) (15Tamburro D. Bratulic S. Abou Shameh S. Soni N.K. Bacconi A. Maccari F. Galeotti F. Mattsson K. Volpi N. Nielsen J. Gatto F. Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans.J. Chromatogr. B. 2021; 1177: 122761Google Scholar) to analyze the urine and plasma protein-free fraction of GAGomes (or free GAGomes, in short). We analyzed the free GAGomes in a good laboratory practice (GLP)-compliant blinded central laboratory in two prospectively enrolled independent cohorts of 308 self-reported healthy subjects with comprehensive demographic and blood chemistry data. We first determined the correlation of free GAGomes with demographic and blood chemistry variables. Next, we established reference intervals for the normal free GAGome in urine and plasma according to accepted guidelines (CLSI EP28-A3c). Finally, we validated the proposed references intervals by transference analysis on two independent cohorts consisting of a total of 140 healthy individuals from two distinct geographical sites. We prospectively enrolled two cohorts of self-rated healthy adults with no history nor family history of cancer (except nonmelanoma skin cancer) from one site in Stockholm, Sweden (Cohort 1, N = 292 and 2, N = 16), for a total of 308 participants (Table 1). Cohort 1 and 2 formed the reference sample group to establish reference intervals.Table 1Subject characteristics in the reference sample group (Cohort 1 and Cohort 2)CharacteristicCohort 1Cohort 2Cohort 1 + 2N29216308Age57 (22–78)43 (27–51)57 (22–78)Gender Female1835188 Male10911120Self-rated health Moderate10010 Good1535158 Very good12911140Blood chemistry biomarkers ALAT (μkat/l)0.36 (0.13–2.44)0.49 (0.27–1.1)0.37 (0.13–2.44) ASAT (μkat/l)0.41 (0.22–1.65)0.41 (0.27–0.59)0.41 (0.22–1.65) Calcium (mmol/l)2.39 (2.15–2.72)2.42 (2.17–2.58)2.39 (2.15–2.72) Creatinine (μmol/l)69 (46–167)80 (59–102)69.5 (46–167) C-reactive protein (mg/l)1.4 (0.2–54.1)0.65 (0.27–5.5)1.3 (0.2–54.1) Estimated glomerular filtration rate (ml/min/1.73 m2)82.5 (33–90)90 (76–90)83 (33–90) Glycated hemoglobin (mmol/mol)-33.5 (28–39)33.5 (28–39) HDL (mg/dl)61.87 (29.78–123.74)-61.87 (29.78–123.74) LDL (mg/dl)131.48 (46.4–239.75)-131.48 (46.4–239.75) Potassium (mmol/l)4.2 (3.5–5.2)4.2 (3.9–4.9)4.2 (3.5–5.2) Prostate specific antigen level (ng/ml)0.8 (0.11–9.8)0.7 (0.33–1.4)0.8 (0.11–9.8) Sodium (mmol/l)139 (132–145)140.5 (138–144)139 (132–145)Complete blood hemoglobin hemoglobin concentration chemistry are as and in in a are as and in characteristics between cohorts (Table 1). the with and all subjects self-reported good or very good health The panel of blood chemistry in Cohort 1 and 2 the for in Cohort 1 and hemoglobin in Cohort 2 blood counts for Cohort 1, Cohort 2 measurements for and chemistry normal for all biomarkers in of the the reference intervals for up to 2 biomarkers in of the and more two in subjects The blood chemistry of C-reactive protein glomerular filtration rate and antigen We subjects with in the reference sample group and performed a analysis of the reference within this The free GAGomes in plasma and urine in a blinded laboratory using a standardized kit on in all Cohort 1 and 2 subjects = We to blood chemistry biomarkers with the normal free GAGome in plasma or urine. we subjects in of their health on the of for the blood chemistry biomarkers we the subjects three no N = one or two N = or more two N = We not between GAGome with of (Table we linear correlations between the concentration of each detectable GAGome and each of the 25 blood chemistry biomarker level a and on correlations that for In we observed weak to correlations = between GAGome features with In the urine concentration with and erythrocyte = and with HDL = In the total and with hemoglobin and erythrocyte = In plasma with = with HDL = We to the free GAGomes in urine and plasma with and that the reference We not between a and GAGome (Table and We observed between and detectable GAGome features 1 and In the of limited to in total and of concentration in males. The urine of on to higher concentration for the disaccharides and resulting in in total and and higher concentration for the HS disaccharides and resulting in in total free chondroitin sulfate (CS) concentration and disaccharide concentration and composition mass fraction in the plasma of the reference sample group (Cohort 1 and HS in plasma. The 1 – - heparan the of on the we to reference intervals by We reference intervals for the free GAGome in urine and plasma healthy and between the of and we established reference intervals for the free GAGome in urine and each according to a and of in in in in We established the reference intervals by of all free and HS features in urine 1 and intervals of total free chondroitin sulfate (CS) and heparan sulfate (HS) concentration and disaccharide concentration and composition in urine by = = HS HS HS HS in a The total free concentration in urine in and in The composition in and The three disaccharides up and and and and of the fraction in urine of and Of the the and to the fraction in and and The urine for and The total free HS concentration in urine in and in The detectable free HS disaccharides HS and The HS urine composition HS and HS in and HS and HS in The HS in and in We the for the free GAGome in plasma of in or in 2 and The free HS fraction in plasma total HS and we from further intervals of total free chondroitin sulfate (CS) concentration and disaccharide concentration and composition in plasma by = = HS in plasma. in a HS in plasma. The total free concentration in plasma in and in The composition and and and (Table for and The free disaccharides The plasma in and in We validated the transference of the established reference intervals for each GAGome in two independent from two distinct geographical (Cohort 3 and by the free GAGomes in urine and plasma in Cohort 3 = and and Cohort = and The for Cohort 3 and for Cohort We observed that Cohort 3 and GAGome we to out the transference analysis in a group Cohort 3 and Cohort by = and and for transference on all free GAGome features in we to = in and to = in males. all free GAGome features in we to = in and to = in males. We determined the of the established reference for each urine and plasma GAGome where for transference In we observed that the transference of reference intervals validated in for the concentration of all detectable free GAGome features (Table The total urine the reference in all total HS within the reference As we observed a a higher in reference sample in in in transference in in and composition for the of reference intervals of free chondroitin sulfate (CS) and heparan sulfate (HS) in urine in independent 3 and = = reference reference HS HS HS HS HS in a In we not the transference of reference intervals for of the two detectable free GAGome features in plasma and to of the transference group concentration (Table The for where to the reference intervals.Table of reference intervals of free chondroitin sulfate (CS) in plasma in independent 3 and = = reference reference in a In this study, we established reference intervals for the free GAGome in urine and plasma in a healthy by advantage of a standardized method (15Tamburro D. Bratulic S. Abou Shameh S. Soni N.K. Bacconi A. Maccari F. Galeotti F. Mattsson K. Volpi N. Nielsen J. Gatto F. Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans.J. Chromatogr. B. 2021; 1177: 122761Google Scholar). In the demographic and of subjects to correlations with the free GAGome. We found that no free GAGome with to in this in urine with total as to free GAGomes glycosaminoglycans in a reference of and postmenopausal Med. Metab. Scholar), in where with reported in glycosaminoglycans in the plasma of of and gender, and to plasma A Med. Metab. Scholar). In to we found that the of several and HS disaccharides higher in with a in urine plasma. in total GAGomes, in the for urine and plasma glycosaminoglycans in a reference of and postmenopausal Med. Metab. Scholar, glycosaminoglycans in the plasma of of and gender, and to plasma A Med. Metab. Scholar). The of to be but we that be attributed to the on the total the free GAGome fraction in as as analytical techniques for GAG measurements and sample We also observed that free GAGomes in urine independent of blood chemistry biomarkers, including of and functions that independent physiological in However, be that and of GAGs are to role in biological such as - as in the of and I. M. D. N.K. and biological functions in and J. Scholar), as as HS and Miller J. glycosaminoglycan and sulfation are for cell of and Chem. 2018; Scholar, Y. Liu Y. W. D. J.J. of glycosaminoglycans in human Rep. 2013; Scholar). The analytical method used not measurements on and which the lack of correlation for The blood chemistry biomarker that with free GAGomes, and urine GAGomes, We that this correlation that GAGomes on the of urine of renal we that by urinary creatinine free GAGome measurements in urine more In free weak correlations with to has been for where their can to of Nilsson M. D. Nilsson B. of chondroitin sulfate A to the of to Scholar), but not in the of blood such as We observed that the reference intervals established for free GAGomes in the reference sample In each free GAGome by most from the in the each by a of from the These that free GAGomes in urine and plasma have and in a healthy by the transference analysis that validated all reference intervals established for urine in independent from two different geographical sites. the free GAGome features that to the reference in the from the reference sample group the plasma concentration in the reference sample group and in the transference that is In the reference for the in the reference sample group to a between the reference we that a of a free GAGome from the reported reference is observed in this be more of a physiological free GAGomes for biomarker In this study established and validated reference intervals for free GAGomes in urine and plasma in the to As we that this study represents a critical resource for physiology and biomarker using free GAGomes in study and in with the and in the The of prospectively with a for reported by the of The study by the in Sweden and study prospectively enrolled self-rated healthy subjects in one site in Sweden two independent Cohort 1 and 2 used to the reference sample group and enrolled between and The as adults between and self-rated no history of cancer (except skin no family history of cancer to The as value in the participants on a by in each formed a and in one also by from participants in Cohort 1 and 2 to laboratory biomarkers of the health of the including the blood the concentration of and C-reactive hemoglobin (Cohort 2 and HDL (Cohort 1 The subjects with referred for clinical but in the The transference analysis performed on two cohorts 3 and of healthy adults from two distinct and geographical sites. Cohort 3 used from the Lifelines Cohort study S. N. M.A. A. F. C. Cohort a study and J. 2015; Scholar). The as adults self-reported The as of cancer within from Lifelines is a study in a the health and of in the of the a of in the and which to the health and of the with a on and complex Cohort enrolled prospectively The as adults self-reported The as in a in a and to in Cohort 1 and in Cohort within The be for to a in Cohort 1 and in Cohort performed the as performed in a blinded laboratory using the Glycosaminoglycan to GAGs from urine and plasma Glycosaminoglycan and in with the for in of the kit (15Tamburro D. Bratulic S. Abou Shameh S. Soni N.K. Bacconi A. Maccari F. Galeotti F. Mattsson K. Volpi N. Nielsen J. Gatto F. Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans.J. Chromatogr. B. 2021; 1177: 122761Google Scholar). the method on the of the protein-free fraction of GAGs disaccharides using and N. Galeotti F. Yang B. Linhardt R.J. Analysis of glycosaminoglycan-derived, precolumn, 2-aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection.Nat. Protoc. 2014; 9: 541-558Google Scholar). The disaccharides using and ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry for and The of the disaccharides using with all as kit (15Tamburro D. Bratulic S. Abou Shameh S. Soni N.K. Bacconi A. Maccari F. Galeotti F. Mattsson K. Volpi N. Nielsen J. Gatto F. Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans.J. Chromatogr. B. 2021; 1177: 122761Google Scholar). measurements of the protein-free fraction of GAGomes the concentration in of resulting in we disaccharides and and HS disaccharides to different sulfation of and HS and one A GAGome detectable in a or its concentration all (15Tamburro D. Bratulic S. Abou Shameh S. Soni N.K. Bacconi A. Maccari F. Galeotti F. Mattsson K. Volpi N. Nielsen J. Gatto F. Analytical performance of a standardized kit for mass spectrometry-based measurements of human glycosaminoglycans.J. Chromatogr. B. 2021; 1177: 122761Google Scholar). The detectable free GAGome further used to including the total of and The concentration in of total and HS as the of the of the and HS we the and HS composition mass of detectable and HS and the and HS the of all sulfated or HS disaccharides by their to all or HS disaccharides by their all GAGome features using the to and intervals established for each free GAGome in urine and plasma using a method The and reference for each GAGome as the and of the of in the reference The correlation between each GAGome and each clinical or variable by linear of a GAGome as variable on a clinical or variable as We the of each correlation using a using the or on clinical or with and as carried out in The data used in this study are not clinical by for to data can be to the The the of with the of this study J. N. and as in to the biomarkers in this the of J. N. and in which the applications, J. N. of the and in no of The Lifelines has been by the from the of and the of the and the in the of the The to the and and for in the and of clinical data and used in this S. B. and S. B. and A. L. S. A. and S. A. M. and data S. A. and S. A. F. N. M. J. and and F. and N. F. and N. J. N. and J. N. and M. J. and M. J. and J. N. and by the and and the and in by the and and the

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