The function of the inter-alpha-trypsin inhibitors in the development of disease.

Through the formation of covalent connections with hyaluronic acid (HA), the inter-α-trypsin inhibitor (IαI) family collaborates to preserve the stability of the extracellular matrix (ECM). The five distinct homologous heavy chains (ITIH) and one type of light chain make up the IαI family. ITIH alone or in combination with bikunin (BK) has been proven to have important impacts in a number of earlier investigations. This implies that BK and ITIH might be crucial to both physiological and pathological processes. The functions of BK and ITIH in various pathophysiological processes are discussed independently in this paper. In the meanwhile, this study offers suggestions for further research on the roles of BK and ITIH in the course of disease and summarizes the plausible mechanisms of the previous studies.

Biochemical diagnosis of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism, comprising over 160 described diseases to this day. CDG are characterized by a dysfunctional glycosylation process, with molecular defects localized in the cytosol, the endoplasmic reticulum, or the Golgi apparatus. Depending on the CDG, N-glycosylation, O-glycosylation and/or glycosaminoglycan synthesis can be affected. Various proteins, lipids, and glycosylphosphatidylinositol anchors bear glycan chains, with potential impacts on their folding, targeting, secretion, stability, and thus, functionality. Therefore, glycosylation defects can have diverse and serious clinical consequences. CDG patients often present with a non-specific, multisystemic syndrome including neurological involvement, growth delay, hepatopathy and coagulopathy. As CDG are rare diseases, and typically lack distinctive clinical signs, biochemical and genetic testing bear particularly important and complementary diagnostic roles. Here, after a brief introduction on glycosylation and CDG, we review historical and recent findings on CDG biomarkers and associated analytical techniques, with a particular emphasis on those with relevant use in the specialized clinical chemistry laboratory. We provide the reader with insights and methods which may help them properly assist the clinician in navigating the maze of glycosylation disorders.

Major endothelial damage markers identified from hemadsorption filters derived from treated patients with septic shock - endoplasmic reticulum stress and bikunin may play a role.

Introduction: In septic patients the damage of the endothelial barrier is decisive leading to circulatory septic shock with disseminated vascular coagulation, edema and multiorgan failure. Hemadsorption therapy leads to rapid resolution of clinical symptoms. We propose that the isolation of proteins adsorbed to hemadsorption devices contributes to the identification of mediators responsible for endothelial barrier dysfunction. Material and methods: Plasma materials enriched to hemadsorption filters (CytoSorb®) after therapy of patients in septic shock were fractionated and functionally characterized for their effect on cell integrity, viability, proliferation and ROS formation by human endothelial cells. Fractions were further studied for their contents of oxidized nucleic acids as well as peptides and proteins by mass spectrometry. Results: Individual fractions exhibited a strong effect on endothelial cell viability, the endothelial layer morphology, and ROS formation. Fractions with high amounts of DNA and oxidized DNA correlated with ROS formation in the target endothelium. In addition, defined proteins such as defensins (HNP-1), SAA1, CXCL7, and the peptide bikunin were linked to the strongest additive effects in endothelial damage. Conclusion: Our results indicate that hemadsorption is efficient to transiently remove strong endothelial damage mediators from the blood of patients with septic shock, which explains a rapid clinical improvement of inflammation and endothelial function. The current work indicates that a combination of stressors leads to the most detrimental effects. Oxidized ssDNA, likely derived from mitochondria, SAA1, the chemokine CXCL7 and the human neutrophil peptide alpha-defensin 1 (HNP-1) were unique for their significant negative effect on endothelial cell viability. However, the strongest damage effect occurred, when, bikunin - cleaved off from alpha-1-microglobulin was present in high relative amounts (>65%) of protein contents in the most active fraction. Thus, a relevant combination of stressors appears to be removed by hemadsorption therapy which results in fulminant and rapid, though only transient, clinical restitution.

Inter-alpha inhibitor proteins attenuate lipopolysaccharide-induced blood-brain barrier disruption in neonatal mice.

There is a paucity of information regarding efficacious pharmacological neuroprotective strategies to attenuate or reduce brain injury in neonates. Lipopolysaccharide (LPS) disrupts blood-brain barrier (BBB) function in adult rodents and increases inflammation in adults and neonates. Human blood-derived Inter-alpha Inhibitor Proteins (IAIPs) are neuroprotective, improve neonatal survival after LPS, and attenuate LPS-induced disruption of the BBB in adult male mice. We hypothesized that LPS also disrupts the function of the BBB in neonatal mice and that IAIPs attenuate the LPS-induced BBB disruption in male and female neonatal mice. IAIPs were administered to neonatal mice after LPS and BBB permeability quantified with intravenous 14C-sucrose and 99mTc-albumin. Although repeated high doses (3 mg/kg) of LPS in neonates resulted in high mortality rates and a robust increase in BBB permeability, repeated lower doses (1 mg/kg) of LPS resulted in lower mortality rates and disruption of the BBB in both male and female neonates. IAIP treatment attenuated disruption of the BBB similarly to sucrose and albumin after exposure to low-dose LPS in neonatal mice. Exposure to low-dose LPS elevated IAIP concentrations in blood, but it did not appear to increase the systemic levels of Pre-alpha inhibitor (PaI), one of the family members of the IAIPs that contains heavy chain 3. We conclude that IAIPs attenuate LPS-related disruption of the BBB in both male and female neonatal mice.

Bikunin: A Promising Prognostic Biomarker for Acute-on-Chronic Liver Failure in Patients with Viral Hepatitis B.

Purpose: To screen out potential prognostic biomarkers for HBV-related acute-on-chronic liver failure (HBV-ACLF). Patients and Methods: Peripheral blood samples of HBV-ACLF patients (n=56) and normal controls (n=15) from the Affiliated Hospital of Southwest Medical University from January 2021 to April 2022 were collected, 5 normal patients and 10 patients with ACLF were randomly selected for Data independent acquisition (DIA) mass spectrometry analysis, and the potential core proteins were screened out via bioinformatics. All samples were validated by Enzyme linked immunosorbent assays (ELISA) technology, and the survival curve was constructed based on the patient's 90-day survival time. Results: A total of 247 differentially expressed proteins (DEPs) were screened, of which 148 were upregulated and 99 were down-regulated. The DEPs were mainly enriched in high-density lipoprotein particle remodeling, coagulation, and hemostasis and participated in signaling pathways such as cholesterol metabolism, coagulation cascades, and PPAR signaling pathway. Finally, bikunin was selected for further study and validated via the ELISA, compared with the normal group, bikunin was poorly expressed in the HBV-ACLF group, the difference was statistically significant (P < 0.0001), the area under the curve (AUC) for Receiver operating characteristic (ROC) analysis was 0.917. Furthermore, compared with the non-survival group, bikunin was highly expressed in the HBV-ACLF survival group, the difference was statistically significant (P=0.0015), and the survival curve showed a positive correlation with patient survival (P=0.0063). Conclusion: The level of plasma bikunin in HBV-ACLF is down-regulated, which is positively correlated with the survival of the patients with HBV-ACLF, and is expected to become a new prognostic biomarker.

Molecular and pathobiological insights of bikunin/UTI in cancer.

Bikunin is a small chondroitin sulfate proteoglycan (PG) with Ser-protease inhibitory activity that plays pleiotropic roles in health and disease. It is involved in several physiological processes including stabilization of the extracellular matrix (ECM) of connective tissues and key reproductive events. Bikunin is also implicated in both acute and chronic inflammatory conditions and represents a non-invasive circulating and/or urinary (as Urinary Trypsin Inhibitor or UTI) biomarker. It exerts inhibitory effects on urokinase-type plasminogen activator (uPA) and its receptor (uPAR) mediating tumor invasiveness by a down-regulation of uPA mRNA expression, thus representing an anti-metastatic agent. However, only limited data on its potential as a diagnostic and/or prognostic marker of cancer have been reported so far. Recent technological advances in mass spectrometry-based proteomics have provided researchers with a huge amount of information allowing for large-scale surveys of the cancer proteome. To address such issues, we analyzed bikunin expression data across several types of tumors, by using UALCAN proteogenomic analysis portal. In this article we critically review the roles of bikunin in human pathobiology, with a special focus on its inhibitory effects and mechanisms in cancer aggressiveness as well as its significance as cancer circulating biomarker.

Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for ß3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome.

The spondylodysplastic type of Ehlers-Danlos syndrome (spEDS) is caused by genetic defects in the B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study, we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion-exchange columns, depolymerized with chondroitinase ABC, and analyzed by nLC-MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non-canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, and 59/41. We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.

Inherited Proteoglycan Biosynthesis Defects-Current Laboratory Tools and Bikunin as a Promising Blood Biomarker.

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases.

SLC37A4-CDG: New biochemical insights for an emerging congenital disorder of glycosylation with major coagulopathy.

SLC37A4-CDG is an emerging congenital disorder of glycosylation which is characterized by a dominant inheritance and a major coagulopathy originating from the liver. Recent studies took interest in the biochemical alterations found in this CDG and showed that they consisted of multiple glycosylation abnormalities, which result from mislocalization of the endoplasmic reticulum glucose-6-phosphate transporter and associated Golgi homeostasis defects. In this work, we highlight in six affected individuals abnormal patterns for various serum N-glycoproteins and bikunin proteoglycan isoforms, together with specific alterations of the mass spectra of endoglycosidase H-released serum N-glycans. Collectively, these data complement previous findings, help to better delineate SLC37A4-CDG and could present interest in diagnosing this disease.

Knockout of the radical scavenger α1-microglobulin in mice results in defective bikunin synthesis, endoplasmic reticulum stress and increased body weight.

α1-microglobulin (A1M) is a ubiquitous protein with reductase and radical- and heme-binding properties. The protein is mainly expressed in the liver and encoded by the α1-microglobulin-bikunin precursor (AMBP) gene together with the plasma proteinase inhibitor bikunin. The AMBP polypeptide is translated, glycosylated and the C-terminal bikunin part linked via a chondroitin sulfate glycosaminoglycan chain to one or two heavy chains in the endoplasmic reticulum (ER) and Golgi compartments. After proteolytic cleavage, the A1M protein and complexed bikunin parts are secreted separately. The complete physiological role of A1M, and the reason for the co-synthesis with bikunin, are both still unknown. The aim of this work was to develop an A1M knockout (A1M-KO) mouse model lacking expression of A1M, but with a preserved bikunin expression, and to study the phenotypic traits in these mice, with a focus on hepatic endoplasmic reticulum (ER) function. The bikunin expression was increased in the A1M-KO mouse livers, while the bikunin levels in plasma were decreased, indicating a defective biosynthesis of bikunin. The A1M-KO livers also showed an increased expression of transducers of the unfolded protein response (UPR), indicating an increased ER-stress in the livers. At twelve months of age, the A1M-KO mice also displayed an increased body weight, and an increased liver weight and lipid accumulation. Moreover, the KO mice showed an increased expression of endogenous antioxidants in the liver, but not in the kidneys. Together, these results suggest a physiological role of A1M as a regulator of the intracellular redox environment and more specifically the ER folding and posttranslational modification processes, particularly in the liver.

The Inter-α-Trypsin Inhibitor Family: Versatile Molecules in Biology and Pathology.

Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved over several hundred million years of vertebrate evolution. IαI is a complex containing the proteoglycan bikunin to which heavy chain proteins are covalently attached to the chondroitin sulfate chain. Besides its matrix protective activity through protease inhibitory action, IαI family members interact with extracellular matrix molecules and most notably hyaluronan, inhibit complement, and provide cell regulatory functions. Recent evidence for the diverse roles of the IαI family in both biology and pathology is reviewed and gives insight into their pivotal roles in tissue homeostasis. In addition, the clinical uses of these molecules are explored, such as in the treatment of inflammatory conditions including sepsis and Kawasaki disease, which has recently been associated with severe acute respiratory syndrome coronavirus 2 infection in children.

Serum bikunin isoforms in congenital disorders of glycosylation and linkeropathies.

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro-α-trypsin inhibitor and inter-α-trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western-blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V-ATPase (ATP6V0A2-CDG, CCDC115-CDG, ATP6AP1-CDG), in Golgi manganese homeostasis (TMEM165-CDG) and in the N-acetyl-glucosamine Golgi transport (SLC35A3-CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in-depth characterized by two-dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases.

CDG biochemical screening: Where do we stand?

BACKGROUND: Glycosylation is one of the most complex post-translational modifications of proteins and lipids, notably requiring many glycosyltransferases, glycosidases and sugar transporters encoded by about 1-2% of all human genes. Deleterious variants in any of them may result in improper protein or lipid glycosylation, thus yielding the so-called 'congenital disorders of glycosylation' or CDG. SCOPE OF REVIEW: We first review the current state of knowledge on the common blood and cellular glycoproteins used in the biochemical screening of CDG, as well as the emerging ones for an improved diagnosis. We then provide an overview of the current state-of-the-art methodologies ranging from gel electrophoresis to mass spectrometry to measure improper glycosylation. Finally, we discuss how additional tools such as metabolomics and microfluidics can be added to the current toolbox to better diagnose and delineate CDG. MAJOR CONCLUSIONS: Combining several biochemical indicators and related methods is often required to cope with the large clinical heterogeneity of CDG and establish a definitive diagnosis. GENERAL SIGNIFICANCE: This review aims to critically present current available CDG biochemical biomarkers and dedicated methods in the context of highly diverse glycosylation pathways and related inherited diseases.

Role of the small proteoglycan bikunin in human reproduction.

PURPOSE: Female reproductive events, including ovulation, menstruation, implantation, and delivery, are physiologically characterized by deep tissue remodeling and display hallmark signs of inflammation. This review discusses the pleiotropic roles played by bikunin in human reproduction. METHODS: A comprehensive literature search of the Medline/PubMed database was performed on the following topics: bikunin structure, roles in pathophysiological conditions and involvement in human reproduction, and usefulness as a marker of gestational complications or as a drug to improve pregnancy outcomes. RESULTS: Bikunin is a small chondroitin sulfate proteoglycan found in blood, urine, and amniotic and cerebrospinal fluids, known for its anti-inflammatory and anti-proteolytic activities. Its levels are usually low, but they can increase several-fold in both acute and chronic inflammatory diseases. Bikunin plays key roles in reproductive events, such as cumulus-oocyte complex formation, pregnancy, and delivery. Its levels have been associated with the most common pregnancy complications such as preterm delivery, pre-eclampsia, and gestational diabetes mellitus. Finally, its intravaginal administration has been reported to reduce the risk of preterm delivery and to improve neonatal outcomes. CONCLUSIONS: Because of its pleiotropic roles in several reproductive events and its association with some life-threatening pathological conditions of pregnancy, bikunin may represent a non-invasive marker for improving follow-up and early diagnosis. Studies showing its usefulness as a drug for reducing the risk of preterm delivery and improving neonatal outcomes have yielded interesting results that deserve to be investigated through further research.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.

The Effect of AMBP SNPs, Their Haplotypes, and Gene-Environment Interactions on the Risk of Atherothrombotic Stroke Among the Chinese Population.

Background/Objectives: Ischemic stroke (IS) is a severe and complex disorder with high morbidity and mortality rates and it has been associated with both environmental and genetic predisposing factors. The purpose of this study was to evaluate the association of the alpha-1-microglobulin/bikunin precursor (AMBP) gene polymorphisms with IS and any possible interactions between specific AMBP alleles and traditional risk factors among a Han Chinese cohort. Materials and Methods: We conducted a candidate gene study designed to characterize nine (9) single nucleotide polymorphisms (SNPs) of the AMBP gene among 195 patients with atherothrombotic stroke (ATS) (a major subtype of IS) and 184 nonstroke controls. Allelic and genotypic frequency differences were evaluated using a logistic regression model. False discovery rate (FDR) correction for multiple comparisons was used. The interactional analyses were performed using the multifactor dimensionality reduction test. Results: We found an association between the rs2567698 CC genotype (odds ratio [OR], 95% confidence interval [CI]: 2.176, 1.159-4.086) and the T allele (OR, 95% CI: 0.654, 0.446-0.960) with risk of ATS in men. However, these associations did not survive FDR correction. In haplotype analyses, the GCCCCCCCC haplotype had a higher frequency (OR, 95% CI: 2.191, 1.048-4.580) in ATS in the ≥45 years of age subgroup, whereas the GCCTCCCCC haplotype decreased the risk for ATS (OR, 95% CI: 0.543, 0.345-0.853) in men. In addition, we also found interactions for ATS risk between SNPs in the AMBP gene and modifiable risk factors for ATS, including: rs11788411 and hypertension in the overall population and women; rs2251680 and hypertension in subjects aged 45 years and older, as well as the interaction among hypertension and the rs2567698 and rs10817564 genotypes in men. Conclusion: Our results show a possible association between AMBP SNP haplotypes and gene-environment interactions with ATS susceptibility in a Han Chinese cohort.

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface.

AIM: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. METHODS: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. RESULTS: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. DISCUSSION/CONCLUSIONS: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

Potential of Protein-based Anti-metastatic Therapy with Serpins and Inter α-Trypsin Inhibitors.

In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.

[Changes in the concentration of urolitiasis markers depending on stone-forming activity in patients with recurrent urolithiasis].

The study aimed to investigate the changes in the concentration of bikunin, osteopontin, and nephrocalcin, depending on the changes in the renal stone-forming activity in patients with recurrent urolithiasis. MATERIALS AND METHODS: The study comprised 152 patients with recurrent calcium oxalate stones at various localizations. Patients of the study group (n=78) were administered complex preventive treatment (water load, Blamaren, thiazide diuretics, oral calcium supplementation) aimed at reducing the activity of urolithiasis. Patients of the control group (n=74) received no treatment. The studied parameters included concentrations of urine bikunin, osteopontin, and nephrocalcin in using ELISA. The follow-up period was six months. RESULTS: By the end of the follow-up, the bikunin concentration in the control group was significantly higher than in the study group (7.0+/-0.81 mg/ml vs. 3.28+/-0.86 mg/ml, respectively, p<0.05) while osteopontin level was significantly lower (2.4+/-0.39 mg/ml vs. 3.4+/-0.36 mg/ml, p<0.05). The nephrocalcin concentrations during the follow-up period did not change significantly (p>0.05). The presence of hypercalciuria did not lead to significant changes in the concentration of stone formation inhibitors. DISCUSSION: The increase in bikunin concentration in control patients is associated with an increase in the expression of this stone formation inhibitor due to the rise in the urolithiasis activity. Reduction in the osteopontin concentration in patients with high urolithiasis activity is a consequence of osteopontin being a constituent of calcium oxalate stones. CONCLUSION: In patients with calcium oxalate urolithiasis, testing for urine concentrations of bikunin and osteopontin as potential markers can be used to estimate the risk of stone recurrence.