Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium–formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.     

Viscosity of Equine Synovial Fluid

Synovial fluid samples from 51 light horses were examined with respect to their rhéologie properties. The analyses were made with a Rotovisco RV3 rotational viscosimeter. Samples from carpal, stifle and hock joints and from healthy joints, joints with synovitis and joints with infectious arthritis were studied. The analyses showed that synovial fluids from both healthy and diseased joints have complex rhéologie properties. In most samples the viscosity varied with the shear rates, the main exceptions being synovial fluids from joints with infectious arthritis. Flow curves (flow behaviour), rather than single observations on viscosity, were considered to be representative for the synovial fluids examined.     

Rheological properties of synovial fluids

Synovial fluid is the joint lubricant and shock absorber [Semin. Arthritis Rheum. 32 (2002), 10-37] as well as the source of nutrition for articular cartilage. The purpose of the present paper is to provide a comprehensive review of the rheological properties of synovial fluid as they relate to its chemical composition. Given its importance in the rheology of synovial fluid, an overview of the structure and rheology of HA (hyaluronic acid) is presented first. The rheology of synovial fluids is discussed in detail, with a focus on the possible diagnosis of joint pathology based on the observed differences in rheological parameters and trends. The deterioration of viscoelastic properties of synovial fluid in pathological states due to effects of HA concentration and molecular weight is further described. Recent findings pertaining to the composition and rheology of periprosthetic fluid, the fluid that bathes prosthetic joints in vivo are reported.     

Evaluation of total LDH and its isoenzymes as markers in preeclampsia

BackgroundPreeclampsia, a rapidly progressing pregnancy-specific multi-systemic syndrome is globally the leading cause of maternal and neonatal morbidity and mortality. This study aims to evaluate the serum total Lactate dehydrogenase levels in women with preeclampsia when compared to normotensive pregnant women and assess the electrophoretic pattern of the LDH isoenzymes in normal pregnancy, preeclampsia and eclampsia.MethodsThe study, carried out in the Department of Biochemistry of MVJ Medical College, included 30 patients of preeclampsia and 30 normotensive gestational age-matched pregnant women admitted to the Department of OBG. Serum total LDH was analysed by DGKC method. Serum and cord blood samples for isoenzyme distribution analysis were collected from a normal pregnant woman undergoing delivery, a woman with mild eclampsia, two women with eclampsia, and analysed by slab gel electrophoresis followed by activity staining.ResultsLDH was significantly elevated in cases as well as between the case (mild and severe) groups, showed a moderate positive statistically significant correlation with systolic, diastolic blood pressure and a sensitivity of 50% and a specificity of 80%. Further, the isoenzyme pattern showed a decreasing distribution of aerobic forms of LDH in preeclampsia-eclampsia.ConclusionsSerum total LDH may serve as a robust and affordable marker of preeclampsia. Serum total LDH, along with its isoenzyme profile, might serve as a predictor and a stronger marker of preeclampsia when compared to serum LDH analysis alone. It may also be used to assess the severity of preeclampsia and hence help in predicting and preventing adverse maternal and foetal outcomes.     

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.     

Unstable Stifles without Clinical or Radiographic Osteoarthritis in Young Goats: An Experimental Study

Thirteen young, castrated male goats had instability of one stifle (knee joint) created by surgical transection of the cranial cruciate ligament, but did not develop any signs of osteoarthritis (OA) in treated joints when confined in limited space for 8 months. At the end of the experiment, the instability in the stifles had not improved, the joints were normal at radiographic examination, there were no signs of inflammation in the synovial membrane or joint capsule, and fibrosis in these tissues was not evident. The articular cartilage was normal both visually and histologically. This may indicate that the young age of the goats and the restricted physical activity on soft floor had prevented the expected development of OA in the experimantally operated joints. Synovial fluid volumes and proteoglycan concentration were measured in the treated and control joints in 6 of the goats. There seemed to be increased quantity of the proteoglycan aggrecan in the synovial fluid from the treated joints compared to the contralateral joints throughout the course of this study. It was concluded that the turnover of aggrecan in the articular cartilage of the treated joints may have been increased.     

LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax

The lactate dehydrogenase isoenzyme patterns have been studied in the axial muscles of the sharks Etmopterus and Galeus. Samples from red, intermediate and white muscle fibres were run separately on a polyacrylamide slab-gel. Both sharks have three isoenzymes; all three are present in the red and intermediate fibres, while the white fibres contain only the two slowest-moving isoenzymes. The red fibres of both sharks contain most of the fastest-moving isoenzyme.
The isoenzymes have a high tolerance towards urea; the slow moving isoenzyme is inhibited at about 2 m urea, the next isoenzyme at 4-6 M urea, and some activity of the fast-moving isoenzyme is still present at 10 M urea in the incubation medium. The LDH distribution in the fibre types is studied by histochemistry on frozen sections.     

Pyroptosis by NLRP3/caspase-1/gasdermin-D pathway in synovial tissues of rheumatoid arthritis patients

We investigated the potential involvement of pyroptosis, a proinflammatory form of regulated cell death, in rheumatoid arthritis (RA). Synovial fluid, synovial tissues and/or serum were compared among 32 patients with RA, 46 patients with osteoarthritis (OA) and 30 healthy controls. Samples were assayed for interleukin (IL)-1β, IL-18 and lactate hydrogenase (LDH). Synovial expression of NLRP3, caspase-1 and cleaved gasdermin D (GSDMD) was assayed using immunohistochemistry and multiplex immunohistochemistry. Patients with RA showed significantly higher levels of IL-1β and IL-18 in synovial fluid than patients with OA, and significantly higher levels of both cytokines in serum than healthy controls. RA was associated with higher levels of LDH in synovial fluid than OA. Among patients with RA, levels of IL-1β, IL-18 and LDH were significantly higher in synovial fluid than in serum, and the levels in synovial fluid positively correlated with disease activity and inflammation. Synovial cells, particularly macrophages, showed upregulation of NLRP3, caspase-1 and cleaved GSDMD in RA compared to OA. Our results implicate pyroptosis in the pathogenesis of RA, perhaps as a driver of local inflammation in joints.     

LDH isoenzyme pattern, lactate/pyruvate, and ultrastructure of fibrinogenic and epithelial cell lines exposed to 3% oxygen

Human diploid fibroblast WI38 and canine epithelial MDCK cells were incubated in an environment flushed continuously for 96 h with 3% and 20% oxygen. Lactate and pyruvate formation and LDH isoenzyme patterns of cells in logarithmic and stationary phases showed characteristic differences between both cell lines. Lactate formation by WI38 cells at 3% oxygen was considerable within the first 18 h. The MDCK cells responded with an even greater lactate formation only after 18 h.The dynamics of LDH isoenzyme changes measured as H/M subunits revealed for WI38 cells a sharp decline after 40 h at 3% oxygen in both logarithmic and confluent stages. MDCK cells show a sharp decline in H/M during the first 40 h (confluent phase) or 20 h (log phase). No changes in either cell line, regardless of the oxygen environment, were seen in the ultrastructure of cells, as seen by transmission electron microscopy. It is suggested that the fibroblast, being better equipped for anaerobic metabolism, quickly produces more lactate without changing the LDH isoenzyme pattern at 3% oxygen. Epithelial cells react first with a modification in the LDH pattern and then a continuous increase in the formation of lactate to very high values.     

Enzymatic activity and distribution of phospholipase A2 in human cartilage

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.     

Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb’s binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 μl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.     

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.     

Comparative assessment of articular cartilage and synovial membrane in experimental haemarthrosis

The changes in articular cartilage and synovial membrane of the knee joints were studied in two groups of rabbits and Wistar rats with experimental haemarthrosis, electron microscopically. Hamarthrosis was produced in group 1 by a single autologous blood injection, in group 2 by intraarticular fracture of the femoral condyles. Samples were taken from the intact articular cartilage, the menisci and the infrapatellar portion of the synovial membrane 12 h to 20 days after intervention. Blood resorption occurs only in the synovial membrane. Fragmentation of erythrocytes and erythrocytophagy by synovial macrophages is documented. The different stages of intracellular digestion of erythrocyte fragments are traced down. Synovial fibroblasts do not participate in erythrocytophagy, although they disclose morphological signs of enhanced functional activity. The findings show changes in the matrix and chondrocytes within the articular cartilage and menisci, and presence of free erythrocytes and lipoprotein complexes amidst the collagen fibres of the matrix. The chondrocytes are poor in cell organelles, while the intracytoplasmic filaments, lipid droplets and glycogen granules are augmented in number. There is no evidence of erythrocytophagy by cartilage cells. On single blood injection in the joint, the ensuing changes are reversible, and the normal synovial membrane structure is restored much quicker than the articular cartilage.     

Protective effects of antioxidants on age-related changes in the electrophoretic patterns of cardiac LDH,hepatic ALP and serum proteins in male golden hamster

Basal and antioxidant-induced changes in the isoenzyme and isoform patterns of cardiac lactate dehydrogenase (EC 1.1.1.27) and hepatic alkaline phosphatase (EC 3.1.3.1), respectively, as well as the electrophoretic patterns of serum proteins in different age groups of male golden hamster were compared. This is to test whether age-induced changes could be corrected by long-term administration of antioxidants. Data indicated that aging causes no remarkable change in the total activity of either cardiac LDH or hepatic ALP, however a significant increase in the fractional activity of some cardiac LDH isoenzymes and a significant reduction in the fractional activity of some hepatic ALP isoforms were induced by aging. On the other hand, long-term administration of antioxidants appeared to manifest a clear counteracting effect on the age-related changes in old age. This effect was indicated in the fractional activity of cardiac LDH isoenzymes and of hepatic ALP isoforms. The present study has also shown a wide-range variation in serum protein patterns due to aging and/or antioxidant administration, which indirectly reflect a parallel variation in the process of gene expression and/or proteolytic activity.     

瘦素对骨关节炎影响的研究进展

瘦素(Leptin)是脂肪因子的一种,可以在骨关节炎(OA)患者的血浆、滑液、软骨中被检测到。OA是一种最常见的关节疾病,其可以发生于全身的多个关节,以骨质和滑膜组织新陈代谢的改变、关节软骨的破坏和由此引起的关节软骨的减少为特征。瘦素是一种由肥胖(0b)基因编码的一个小的非糖基化肽激素。最开始,瘦素仅仅被认为是一种脂肪细胞源性的小分子(16KD),在下丘脑中枢水平作为一个饱感因子介导食物摄入量减少,并增加能量的消耗。现在已经发现,瘦素在机体内可发挥多种生理作用,并与OA病情有关。本文通过对瘦素与OA、软骨、肥胖、生物标志物、脂联素等之间的联系做一综述,以了解瘦素与OA之间的联系,为OA的治疗方面的进一步研究提供帮助。     

Diagnosis of rheumatoid arthritis of knee joints using magnetic resonance imaging]

A combination of clinical, X-Ray and magnetic resonance tomographic studies for 129 knee joints was made in 85 patients with rheumatoid arthritis of knee joints. MRI symptoms of rheumatoid arthritis of knee joints, including fluid accumulation in the articular cavity, degeneration of the articular cartilage, meniscus, ligaments, proliferation of the synovial membrane, destructive changes in osseous epiphysis were defined. Comparative analysis of the X-Ray and MRI imaging findings has shown that MRI has advantages structures of joints in rheumatoid arthritis.     

Growth factors with heparin binding affinity in human synovial fluid

Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of 3H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.     

Biochemical purification of monkey and human LDH isoenzyme 5 suitable as immunization antigen

A method consisting in an ion-exchange chromatography on DEAE-Sephadex A50 and an affinity chromatography on Oxamate-hexyl-Sepharose for the purification of monkey and human LDH isoenzyme 5 is presented. Analytical isoelectric focusing reveals minor variants in the monkey LDH isoenzyme 5. This material has been used to produce an antiserum from rabbits which recognizes all the human LDH isoenzymes containing the M subunit, without cross-reacting with LDH1.     

Lactate dehydrogenase isoenzyme pattern and total LDH activity in the lung and liver of rats chronically exposed to low and high oxygen concentrations.

Young adult rats were continuously exposed for 44 and 84 days to environments containing 9–11%, 20%, and 80% oxygen. Low and high oxygen atmospheres were achieved by using boxes laminated with silicone rubber membranes which have a differing permeability for oxygen on one side and for carbon dioxide and nitrogen on the other. Animals exposed to both extreme concentrations significantly slowed body growth, and the weight of the lungs was proportionally less. The pattern of LDH isoenzymes in the lung showed the presence of all five characteristically changing isoenzymes in relation to oxygen concentration. A marked increase of M subunits in the LDH in lungs of rats exposed to low oxygen indicated a higher tissue concentration of lactate. Thus, the percentage of M subunits was significantly higher in low oxygen and significantly lower in lungs of rats exposed to high oxygen as compared to controls. Under the same experimental conditions there were no changes in the liver LDH isoenzyme pattern. Total LDH activity in the lungs of rats exposed to either extreme oxygen atmosphere was significantly elevated as compared to controls kept at an ambient atmosphere. It is concluded that chronic exposure of rats to low as well as to high oxygen was injurious to the lung tissue, as evidenced by total LDH activity. Thus, LDH isoenzyme pattern in the lung reflected the actual gas exposure (pO₂), rather than local tissue metabolism.     

Additional lactate dehydrogenase (LDH) isoenzymes in normal testis and spermatozoa of adult man

Summary Adult human testicular tissue contains up to six previously undescribed lactate dehydrogenase (LDH) isoenzymes in addition to the five LDH isoenzymes normally found and the sixth found in spermatogenic cells and spermatozoa, LDH-X. Additional LDH isoenzymes were also found in spermatozoa but not in seminal fluid or in serum. After electrophoresis one additional LDH isoenzyme of testicular tissue was localized between LDH-1 and LDH-2, two between LDH-2 and LDH-3, two between LDH-3 and LDH-4, and two between LDH-4 and LDH-5. These localizations indicate that the additional LDH isoenzymes are tetramers combining the A and B subunits of the five normal LDH isoenzymes and the C subunit of LDH-X. The additional LDH isoenzymes may be important in the metabolism of spermatogenic germ cells and spermatozoa.