Janusz Popko

Optimization of an enzymatic method for the determination of lysosomal N-acetyl-β-D-hexosaminidase and β-glucuronidase in synovial fluid

Abstract Background: Our goal was to develop a suitably sensitive assay for N-acetyl-β-D-hexosaminidase (HEX) and β-glucuronidase to allow their use as markers of joint diseases. Methods: We optimized a spectrophotometric method for the determination of lysosomally derived HEX and β-glucuronidase in synovial fluid on a microplate reader to improve its utility. HEX and β-glucuronidase act on the 4-nitrophenyl derivatives N-acetyl-β-glucosamine and β-D-glucuronide, respectively, to produce 4-nitrophenol, which can be measured at 405nm on a microplate reader. Results: Maximum enzyme activity was observed at pH 4.7 in a citrate-phosphate buffer for HEX and at pH 4.5 in an acetate buffer for β-glucuronidase. A 10-μL sample with 30μL of substrate solution and 40μL of appropriate buffer produced measurable amounts of 4-nitrophenol after incubation for 60min at 37°C. Reactions were terminated by the addition of 200μL of 200mM borate buffer (pH 9.8). Conclusions: The assay is sufficiently sensitive for small volumes of synovial fluid, and is useful for the clinical diagnosis of joint diseases. Clin Chem Lab Med 2006;44:933–7.

Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis

Lysosomal exoglycosidases participate in the destruction of the articular cartilage by cleaving glycoside bonds in glycoproteins and proteoglycans. The aim of the study was to determine the activity of exoglycosidases: hexosaminidase, β-glucuronidase, β-galactosidase, α-mannosidase and α-fucosidase in serum and synovial fluid of patients with Lyme and rheumatoid arthritis. The study group consisted of 10 patients with chronic Lyme arthritis (age 18 – 74 y), 13 with rheumatoid arthritis (age 32 – 70 y) and 10 with juvenile idiopathic arthritis (age 8 – 17 y). The control group consisted of 9 healthy volunteers (age 24 – 62 y). The activity of the exoglycosidases was determined with the p-nitrophenyl derivatives of sugars as substrates. A significant increase of the activity of all the exoglycosidases in serum and in synovial fluid of the patients with different forms of arthritis was found. The ratio of synovial fluid/serum activity of exoglycosidases was above 2.0 in LA but not in JIA and RA patients. As the main source of exoglycosidases in the joint is the synovial membrane, this result supports the appropriateness of therapeutic synovectomy in chronic Lyme arthritis with knee effusion. The serum activity of hexosaminidase may be used in monitoring the course of Lyme arthritis and the efficiency of treatment.

Association between Vitamin D Receptor Polymorphism and Serum Vitamin D Levels in Children with Low-Energy Fractures.

Objective: Fractures of bones, especially forearm fractures, are very common in children and their number is increasing. This study was designed to determine the impact of vitamin D serum levels and vitamin D receptor (VDR) polymorphisms on the occurrence of low-energy fractures in children. Methods: The study group consisted of 100 children with clinically relevant bone fractures and a control group consisted of 127 children without fractures. Total vitamin D [25(OH)D3 plus 25(OH)D2] serum concentrations were evaluated in every patient. Genotypes for 4 restriction fragment length polymorphisms of the vitamin D receptor gene (FokI, ApaI, TaqI, and BsmI) were determined by standard polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) techniques. Results: Differences in concentrations of vitamin D were observed between the group with bone fractures (median = 12 ng/ml) and the control group (median = 16 ng/ml; p = 0.000044). Higher levels of vitamin D reduced the risk of fracture by 1.06 times (p = 0.0005). No impact of particular VDR polymorphism on the occurrence of low-energy fractures in children was detected. However, there were significant differences in the prevalence of FokI polymorphism genotypes between the fracture and control groups (p = 0.05). Furthermore, the recessive “aa” genotype of ApaI polymorphism and the dominant “TT” genotype of TaqI polymorphism were associated with higher levels of vitamin D (p = 0.005 and p = 0.036, respectively). Conclusions: Vitamin D deficiency is an independent risk factor for fractures in children. ApaI polymorphism recessive “aa” and TaqI polymorphism dominant “TT” genotypes are associated with higher levels of vitamin D in serum.

Phenotype variability in a daughter and father with mild osteogenesis imperfecta correlated with collagen and prolidase levels in cultured skin fibroblasts.

Studies of collagen biosynthesis and prolidase activity were performed on cultured skin fibroblasts obtained from a female patient and her father, who displayed variable phenotypes of mild osteogenesis imperfecta (OI). For comparison, the same studies were also performed on age-matched controls. Biosynthesis of collagen in fibroblasts of the less affected father was reduced to approximately 50% of control levels, whereas in cells of the more severely affected daughter, it was decreased to about 20% of control levels. Furthermore, the decrease in collagen synthesis in OI fibroblasts was accompanied by a parallel decrease in prolidase activity and expression of beta1 integrin and insulin-like growth factor-I (IGF-I) receptors recovered from the cells. Therefore, prolidase, as well as IGF-I and beta1 integrin receptors involved in collagen metabolism regulation, may represent important factors influencing OI phenotype.

IGF-I and IGF-Binding Proteins in Articular Exudates of Children with Post-Traumatic Knee Damage and Juvenile Idiopathic Arthritis

Objective: IGF-I stimulates multiple functions of connective tissue cells and its activity is modulated by IGF-binding proteins (BPs). Some metalloproteinases are expected to modify IGF-I activity by digestion of IGF-BPs. It was decided to evaluate the concentration of IGF-I, IGF-BPs and the activity of gelatinases A and B in knee exudates of children with post-traumatic damage (PTD) and children with juvenile idiopathic arthritis (JIA) in comparison with those in the sera of the same patients. Methods: ELISA (for IGF-I assay), polyacrylamine gel electrophoresis following Western immunoblotting (for IGF-I and IGF-BPs expression), and zymography (for gelatinase detection) were used. Results: The knee exudates, especially those taken from patients with JIA, contained large amounts of IGF-I. The exudates of PTD and JIA patients contained some forms of IGF-BP-1 of molecular weight lower than those occurring in serum. Low expression BP-3 and high activity of gelatinase B were detected in the JIA exudates. Conclusions: The high gelatinase activities in exudates imply joint tissue damage. The cellular response to damage of this kind is an increase in IGF-I production, which stimulates repair processes. High proteolytic activities of gelatinase B in JIA patients may lower the amount of BP-3, possibly causing a relative decrease of IGF-I concentration and impairing the reparation processes stimulated by IGF-I.

Roles of Vitamins D and K, Nutrition, and Lifestyle in Low-Energy Bone Fractures in Children and Young Adults

ABSTRACT The research on skeletal system health in children and young adults, while recognizing the important role of calcium and vitamin D, goes beyond these nutritional standards. This review focuses on the role of vitamin K in combination with vitamin D and other factors in bone health. The current understanding is that maintaining bone health and prevention of low-energy fractures in any pediatric population includes nutritional factors combined with an active lifestyle. Calcium, vitamin D, and vitamin K supplementation contribute independently and collectively to bone health. The beneficial role of vitamin K, particularly vitamin K2 as menaquinone-7 (MK-7), in bone and cardiovascular health is reasonably well supported scientifically, with several preclinical, epidemiological, and clinical studies published over the last decade. Osteocalcin and matrix-Gla (glutamate-containing) protein (MGP) exemplify vitamin K–dependent proteins involved in building bone matrix and keeping calcium from accumulating in the arterial walls, respectively. An important part of the mechanism of vitamin K involves carboxylation and posttranslational activation of the family of vitamin K–dependent proteins, which prevent expression of pro-inflammatory factors and support improvement in bone mineral concentration, bone mineral density, and the quality of bone matrix. Understanding the combined approach to a healthy skeletal system in children and young adults, including the roles of vitamins D and K, calcium, healthy diet, and exercise, is particularly important in view of reports of subclinical insufficiency of vitamins D and K in otherwise healthy pediatric populations with low-energy bone fractures.

Glycoconjugate markers of joint diseases

A number of different types of glycoconjugate are found associated with joint tissue and fluids, comprising glycoproteins, glycolipids and glycosaminoglycans. Oligosaccharide chains of glycoconjugates are degraded by exoglycosidases, and the dominant exoglycosidase found in human blood, synovial fluid, the synovial membrane and chondrocytes of articular cartilage is HEX (N-acetyl-β-hexosaminidase). HEX is localized mostly intracellularly in synovial cells. Serum activity of HEX may be used to monitor the course and efficiency of treatment of Lyme arthritis, and activity of HEX, above 10 μkat/kg of protein in the synovial fluid, suggests rheumatoid disease. There is a shortage of HEX inhibitors able to penetrate synoviocytes, so the development of drugs which inhibit synthesis and/or the activity of HEX will be a promising field for future investigations.

Differential effect of platelet-rich plasma fractions on β1-integrin signaling, collagen biosynthesis, and prolidase activity in human skin fibroblasts

The study was conducted to evaluate the effects of platelet-rich plasma (PRP), supernatant of PRP (SPRP) obtained by centrifugation, and supernatant of activated PRP (SActi-PRP) obtained by Ca2+ solution-treated PRP on collagen biosynthesis, prolidase activity, and β1-integrin signaling in cultured human skin fibroblasts. Incubation of fibroblasts with 5% PRP for 24 h contributed to ~5-fold increase in collagen biosynthesis compared to the control. In the cells treated with 5% of SPRP or SActi-PRP, collagen biosynthesis showed a 3-fold increase of the control. PRP, SPRP, and SActi-PRP stimulated prolidase activity similar to collagen biosynthesis. Collagen biosynthesis and prolidase activity are regulated by β1-integrin receptor signaling. Incubation of fibroblasts with PRP for 24 h contributed to a dose-dependent increase in the expression of β1-integrin receptor, while SActi-PRP increased the process to a much lower extent. SPRP had no effect on the β1-integrin receptor expression. All the studied fractions of blood increased the expression of FAK as well as the expression of phosphorylated MAP-kinases. However, PRP was found to be the most effective stimulator of expression of these particular kinases. These studies suggest that a complex of factors, including growth factors, adhesion molecules, and prolidase contained in PRP, all evoke growth and collagen-promoting activities in human dermal fibroblasts.

Fibroblast-Like Synovial Cells in Rheumatoid Arthritis - the Impact of Infliximab on Hexosaminidase Activity

BACKGROUND The effect of multiple infusions of infliximab (INF), a chimeric anti-tumor necrosis factor alpha antibody, on the concentration of hexosaminidase (HEX) activity in a synovial cell culture derived from human synovial inflamed fluid obtained from patients suffering from rheumatoid arthritis (RA) has been evaluated. OBJECTIVES The aim of this study was to prove INF efficacy in RA. MATERIAL AND METHODS Inflamed synovial fluid was taken from RA patients (a study group) and patients who had undergone knee trauma within 7 days (a control group). The following solutions of infliximab were used: 40, 60 and 140 µg/mL. Determination of the concentration of HEX activity in cell cultures was performed after 24, 48, 72 and 96 h of infliximab administration. To identify synoviocytes in cell culture immunohistochemical staining with vimentin and pancytokeratin was performed. RESULTS A predominance of fibroblast-like synovial cells has been observed in the study group. In the control group the concentration of HEX activity without adding infliximab to the cell culture was 283.00 nkat/mL. After 96 h of incubation with infliximab, the concentrations of HEX activity in cultured synoviocytes according to infliximab doses of 40, 60 and 140 µg/mL were respectively: 280.00, 271.50 and 293.50 nkat/mL. In the study group, the concentration of HEX activity without adding infliximab to the cell culture was 542.27 nkat/mL. The final concentrations of HEX activity of cultured fibroblast-like synovial cells measured after 96 h of incubation with infliximab were: 471.72, 498.27 and 556.72 nkat/mL, according to infliximab doses of 40, 60 and 140 µg/mL. In all groups (besides the infliximab concentration of 140 µg/mL after 96 h of incubation), the level of concentration of HEX activity was significantly higher in the study group compared to the control group, irrespective of infliximab concentration and time of infliximab incubation. CONCLUSIONS Infliximab changes the concentration of HEX activity depending on the drug dose and time of administration.

Lysosomal Glycosidases in Degradation of Human Articular Cartilage

Joint diseases cause serious medical problems for several million people world-wide and therefore the World Health Organization has designated years 2000-2010 as the Decade of the Bone and Joint (Popko et al.2011). Osteoarthritis (OA) is the most common, and increasingly prevalent, human joint disorder (Dieppe, 2000). It has been estimated that in 1990 12 % of Americans, nearly 21 million people had clinical symptoms of osteoarthritis (Lawrence et al., 1998). Rheumatoid arthritis (RA) affects about 0.3 to 1.5% of the world population(Chikanza et.al., 1998). Juvenile idiopatic arthritis (JIA) is one of the most common rheumatic diseases in children, which causes pain and functional disability. According to a 2008 study performed by the National Arthritis Data Workgroup, there were close to 3000,000 children in the U.S.A. with some form of juvenile arthritis (Giannini et al., 2010). Lyme arthritis (LA) caused by spirochete Borrelia burgdorferi, is increasing in prevalence disease involving the musculoskeletal system, particularly affecting knee joints (Pancewicz et. al.2009). RA and JIA are chronic autoimmune inflammatory diseases primarily affecting the synovial membrane, leading to joint damage and destruction. OA is the most common joint disorder and a major public health problem in western populations (Lawrence et al. 1998). Clinical and epidemiological studies on OA have recognized a series of etiologic factors including local factors (such as malformations or joint injuries) and systemic factors (such as overweight, race, gender, or metabolic diseases). OA is associated with a loss of proper balance between synthesis and degradation of the macromolecules that gives articular cartilage its biomechanical and functional properties. Concomitantly in OA, changes occur in the structure and metabolism of the synovium and subchondral bone of the joint.