Authors:Petra Seebeck, Ph.D.,

1 and Peter Haima, Ph.D.,

2

1. Zurich Center for Integrative Human Phys. ZIHP, Core Facility for Rodent Physiology, University Zurich, Switzerland

2. Life-Force biomedical communication, Netherlands

TECOmedical Clinical & Technical Review

August 2013

Biomarker for liver fibrosis and cirrhosis, joint disease, inflammation and others.

Hyaluronic Acid (Hyaluronan)

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CONTENTS

1 Biochemical&functionalbackground 3

2 Synthesis,turnover&catabolism 4

3 BiomarkerforDiagnosisandmonitoringofdisease 53.1 Liverdisease 5 Liverfibrosisandcirrhosis 6 MonitoringpatientswithchronichepatitisCandB(HCV,HBV). 73.2 Jointdisease 83.3 Malignanttumors 93.4 Diabetes 93.5 Otherindications 9

4 Therapeuticandcosmeticapplicationsofhyaluronicacid 10

5 MeasurementOfHyaluronicAcid 105.1 FactorsAffectingHyaluronicAcidMeasurements 10 Demographicfactors 10 FoodIntake/Diet 11 Physicalactivity 11 Comorbidities 12 Drugs 12 Sampletype 125.2 NormalValuesOfHyaluronicAcid 12 ReferenceValues 12

6 LiteratureReferences 12

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1 BIOCHEMICAL&FUNCTIONALBACKGROUND

Hyaluronic acid (also called Hyaluronan, hyaluronate or HA) is a high molecular weight biopolysaccharide, discovered in 1934 in the vitreous of bovine eyes [74]. The name is derived from “hyalos”, which is the Greek word for glass and uronic acid. It consists of repeated disaccharide units which are composed of D-glucuronic acid and D-N-acetylglu-cosamin (Figure 1) [10]. The number of disaccharides can reach about 10,000 in one molecule, so that hyaluronic acid could possess a molecular weight between 106 and 107 Da and extend up to more than 10 µm (≈ diameter of erythrocyte).

Hyaluronic acid is a major component of connective tissues and thus distributed ubiquitously in the organism. About one-half of the body’s entire hyaluronic acid is found in the skin and about one fourth in the skeleton and its suppor-ting structures like ligaments and joints (Figure 2 [78]). Hyaluronic acid is placed in the areas between cells, where it absorbs and holds water to act as a cushion for the body’s tissues. It is essential for the structure and organization of extracellular matrices since it forms a network interacting with proteins, receptors and cell surfaces.

Hyaluronic acid can trap up to 1000 times its weight in water, giving it stiff viscous quality similar to gelatinous desserts. The biological functions of hyaluronic acid include maintenance of the elastoviscosity of liquid connective tissues as joint synovial and eye vitreous fluid, control of tissue hydration and water transport, supramolecular assem-bly of proteoglycans in the extracellular matrix, and numerous receptor mediated roles in cell detachment, mitosis, migration, tumor development and metastasis, and inflammation. Its main function in the body is to bind water and lubricate movable parts of the body, such as joints and muscles.. Since concentrated solutions of hyaluronic acid pos-sess a shear-dependent viscosity the synovial fluid is able both to resist rapid deformation as well as to allow viscous gliding during slow deformation [42]. Additionally, hyaluronic acid is involved in wound healing and highly expressed in granulation tissue (connective tissue and tiny blood vessels that form on the surfaces of a new wound) [43].

Figure 1: Hyaluronic acid is a large linear non-sulfated glyco-saminoglycan (GAG) consisting of repeated disaccharide units (C14H21NO11)n

Figure 2: The main part of the body’s entire hyaluronic acid is found in the skin and the skele-ton [78].

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2 SYNTHESIS,TURNOVER&CATABOLISM

Hyaluronic acid is synthesized by fibroblasts and other specialized connective tissue cells e.g. synovial lining cells or hepatic stellate cells. It is synthesized by integral membrane proteins called hyaluronic acid synthases of which three different isoformes have been found in vertebrates: HAS1, HAS2, HAS3. The tissue half-life of hyaluronic acid differs between tissue types and species (Figure 3) and varies from about one to several days, e.g. < 1 day for skin and 2 – 3 weeks for cartilage. A human individual of seventy kilograms possesses fifteen grams of hyaluronic acid of which one third is cycled daily [54].

The degradation of hyaluronic acid occurs in a series of discrete steps generating hyaluronic acid chains of decreasing sizes with widely differing biological functions of the oligomers after each degradation step, e.g. low molecular weight hyaluronic acid possesses pro-angiogenic properties whilst certain fragments can induce inflammatory responses in macrophages and dendritic cells. Hyaluronic acid is degraded by a family of enzymes named hyaluronidases. A small amount (10 – 30 %) of the tissues’ high molecular weight hyaluronic acid is partly degraded to lower weight hyaluro-nic acid but the much larger part is removed and degraded by the lymphatic system [9].Adult cartilage is avascular and depends upon the synovial fluid providing its nutrition as well as the disposal of metabolic wastes. Thus, hyaluronic acid resulting from cartilage degradation is firstly released into the synovial fluid from where it enters the lymph stream through the highly vascularized synovial membrane [2]. The remaining low molecular weight fragments enter the blood circulation from where 90 % is cleared by the liver and about 10 % by the kidneys and the spleen (Fig. 4). Therefore, a decrease in liver function has an immediate effect on the serum levels of hyaluronic acid.

The serological half-live of hyaluronic acid is about 2 – 5 minutes [28, 42]. The normal adult human serological level of hyaluronic acid varies between 10 and 100 µg/l and the total turnover of hyaluronic acid in serum is estimated to be in the range of 10 – 100 mg/24 h [27].

Figure 3: Normal values of HA in different animal species. [42]

Figure 4: Path of HA degradation. HA de-rived from cartilage enters the lymph circu-lation via the synovial membrane [29].

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3 BIOMARKERFORDIAGNOSIS&MONITORINGOFDISEASE

Serum levels of Hyaluronic acid (HA) are typically low in healthy individuals as circulating HA is rapidly removed from the circulation by receptor-mediated clearance by liver endothelial cells. Increased HA serum levels can be found either due to a decreased liver function (e.g. liver fibrosis/cirrhosis) or excessive synthesis of hyaluronic acid (e.g. joint or skin disease, cancer). Serum HA levels are therefore extremely useful to diagnose and monitor liver and joint disease and to monitor the efficacy of therapy. Increased serum HA levels are also observed during inflammatory processes like psoriasis or sclerosis and septic conditions and in diabetic patients. Hereditary diseases with disturbances of the metabolism of HA like Werner’s syndrome (Pangeria) and Hutchinson-Gilford progeria syndrome (premature aging) show increased urinary and serum HA levels.

3.1 LIVERDISEASE

As 90% of all serum HA is cleared by the liver, a decrease of liver function is immediately reflected by an increase in serum HA levels. This makes HA an excellent marker to diagnose and monitor liver pathologies. Liver fibrosis, cirrhosis, inflammation and steatosis are major features of acute and chronic liver diseases, such as viral hepatitis (HBV, HCV), autoimmune or metabolic liver diseases, alcoholic or nonalcoholic steatohepatitis ((N)ASH). They are also associated with an increased risk of developing hepatocellular carcinoma. Prediction of fibrosis and steatosis is essential for the management of these patients. Liver fibrosis/cirrhosis is characterized by an enhanced extracellular matrix synthesis by hepatic stellate cells leading to progressive induration of the entire organ (Figure 5). The formation of scar tissue causes the progressive loss of liver function and also decreases the capacity of the liver sinusoidal endothelial cells for the clearance of hyaluronic acid. Serum HA levels increase with the development of liver fibrosis, with highest levels observed in cirrhosis, due to the reduction of HA receptors and increased synthesis of HA by the stellate cells and simultaneous release from the necrotic liver. Liver biopsy is currently the gold standard for the detection of early liver damage. This invasive examination me-thod is limited by sample errors, sample variation and the risk of clinical complications. Conventional markers of liver damage, liver transaminases (AST/ALT), frequently provide incorrect information about liver damage. For example, it could be shown that up to 25–30 % of the patient with fibrosis liver damage have normal transaminase levels [65, 66]. Serum HA would be clinically useful as a non-invasive routine marker of liver fibrosis or cirrhosis that reflects the function of the sinusoidal endothelial cells.

HA has been extensively studied in patients with different causes of liver disease (Fig. 6). It proved to be the best single biomarker of fibrosis and a cost effective and non-invasive way to exclude cirrhosis. Additionally, serum HA was shown to be a sensitive marker for the rejection of liver transplants [41, 48], elevated levels of serum HA within the first 6 months after liver transplantation accurately predicted rapid fibrosis progression [48].

Figure 5: Progression of liver damage leading to liver fibrosis/cirrhosis.

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Figure 6. Diagnostic performance of the biomarker Hyaluronic Acid in patients with different causes of liver disease..

Liverfibrosisandcirrhosis

Serum HA levels increase with the development of liver fibrosis, and correlate with the degree of fibrosis and inflam-mation (Figure 7) [14, 44, 51, 57, 69].

The analysis of serum HA can help to discriminate between insignificant and significant liver fibrosis or to exclude severe fibrosis and cirrhosis as well as to support the monitoring of patients with the risk of progressive fibrosis (figure 8 [14]) [25]. The serological level of HA correlates with histopathological findings obtained by liver biopsy [38, 55]. Further correlations were observed between the serological concentration of hyaluronic acid and parameters of liver function e.g. AST, ALP and GGT [11].

Fig. 7. HA levels in different stages of liver fibrosis (69)

Fig. 8. HA levels in 405 patients with chronic hepatitis were used to prospectively predict significant fibrosis, severe fibrosis, and cirrhosis and predict absence of significant fibrosis, severe fibrosis, and cirrhosis [14].

*NPV: Negative Predicted Value; PPV: Positive Predicted Value

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MonitoringpatientswithchronichepatitisC+B(HCV,HBV)In patients with chronic hepatitis C virus (HCV), HA levels increase with the development of liver fibrosis. Moreover, in patients with cirrhosis, HA levels correlate with clinical severity [70, 71, 72]. Absence as well as presence of significant fibrosis, severe fibrosis, and cirrhosis can be predicted by HA levels [14].Serum hyaluronic acid can also help to monitor antiviral or antifibrotic therapies [40]. In chronic viral hepatitis the level of hyaluronic acid decreased in patients responding to antiviral therapy [12, 30]. In patients with chronic hepatitis C serum hyaluronic acid was used to predict the response to interferon therapy where decreasing levels of hyaluronic acid correlated with the histological improvement of liver tissue [62, 63]. Additionally, the level of serum hyaluronic acid was found to predict the occurrence of severe complications in hepatitis C cirrhosis [13]. In patients infected with hepatitis B the serum level of hyaluronic acid decreased during lamivudine treatment (Figure 10) [11].

HA is a particular useful marker to monitor liver disease in alcoholic patients and to exclude cirrhosis (negative predic-tive value is 100% [14]. Serum HA reflects the severity of liver inflammation, fibrosis, and fibrogenesis in patients with alcoholic liver disease and is useful as a marker of precirrhotic and cirrhotic stages (Figure. 9) [44].

FIG.9. Serum HA levels in alcoholic patients. Shaded area indicates normal values. N, normal liver; FC, fatty changes; F, fibrosis; AH, alcoholic hepatitis; C, cirrhosis; CAH, cirrhosis with superimposed alcoholic hepatitis [44].

Figure 10: In patients with chronic hepatitis B infection lamivudine treatment decre-ased the serological concentration of HA. HA 0 = before treatment, HA I = 4 weeks,HA II = 12 weeks, HA III = 24 weeks, HA IV = 48 weeks of lamivudine treatment (mean ± std.-dev.) [11].

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3.2 JOINTDISEASE

Osteoarthritis (OA) and rheumatoid arthritis (RA) are the most common types of degenerative joint diseases. Chronic osteoarthritis is characterized by the progressive destruction of articular cartilage with concurrent damage of the subchondral bone, whilst in rheumatoid arthritis chronic synovial inflammation with marked proliferation of the synovial membrane leads to thinning out of the articular cartilage . In non-inflammatory cartilage degeneration the progressive breakdown of articular cartilage predominates, incre-asing both the synovial and serological level of HA. In contrast, proliferative synovial inflammation causes a forced synthesis of HA which increases the hyaluronic acid concentrations in synovial fluid and blood (Figure 5). However, joint inflammation might also occur due to joint injury and - at least temporarily - during all types of joint diseases.

In patients with osteoarthritis as well as rheumatoid arthritis the concentration of hyaluronic acid correlated with the degree of joint inflammation and synovial proliferation as well as with the degree of joint space narrowing with a more progressive course of disease observed in patients with higher initial values [20, 26, 37, 45]. In patients with osteoarthritis the concentration of hyaluronic acid correlated with the radiographic signs of osteoarthritis as well as with the length of osteophytes [23].

In patients with rheumatoid arthritis the level of hyaluronic acid correlated with the clinical signs of the disease e.g. joint swelling and morning stiffness. Additionally, those patients showed markedly increased concentrations of hya-luronic acid 0.5 – 2 hours after leaving bed corresponding with a decrease in joint stiffness [33]. Additional informa-tion might be obtained by sampling during the morning hours in these patients. In patients with rheumatoid arthritis, correlations were observed between the serological concentration of hyaluro-nic acid and other inflammation markers like CRP (C-reactive protein), ESR (erythrocyte sedimentation rate) and the connective tissue marker PIIINP (aminoterminal propeptide of type III procollagen) [35, 61]. Anti-inflammatory therapy reducing synovial inflammation in rheumatoid arthritis lowered the level of hyaluronic acid [53]. Decreasing levels of serum hyaluronic acid were also observed in rheumatoid arthritis patients responding to disease modifying antirheumatic drugs (DMARDs) correlating with decreasing levels of inflammation markers and PIIINP and less clinical symptoms (Figure 12) [20, 26, 37, 45].

Figure 11: Increased HA levels might re-sult from cartilage degradation as well as synovial inflammation: HA resulting from cartilage degradation is released into the synovial space from where it enters lymph circulation via the highly vascularized synovial membrane.

Figure 12: Correlation of serum HA levels with clinical signs of synovitis and serolo-gical levels of inflammation markers and PIIINP in patients with RA responding to two years of treatment with DMARDs [20].

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3.3 MALIGNANTTUMORS

HA levels are elevated in various cancer cells and it is believed to form a less dense matrix, enhancing the cancer cell’s motility and invasive ability into other tissues [77]. Also the various types of molecules that interact with HA can contribute to many of the stages of cancer metastasis.Certain malignancies e.g. mesothelioma and Wilms’ tumor are able to produce either HA or factors which affect other cells to produce HA and are thus accompanied by high HA serum levels [17, 59]. In prostate and breast cancer the serological level of hyaluronic acid has been demonstrated to correlate with malignancy [1, 24, 47]. The analysis of the hyaluronic acid blood levels might therefore be used to monitor disease progression as well as to detect the patients’ responding to a therapy. In bladder cancer the synthesis of hyaluronic acid was found to be associated with tumor angiogenesis and metastasis. Increased urinary levels of hyaluronic acid indicated the presence of bladder cancer regardless of tumor grade. The analysis of urine hyaluronic acid – in combination with the measurement of hyaluro-nidase – could therefore be used to detect bladder cancer as well as to monitor patients for residual carcinoma after tumor resection [30, 34].

3.4 DIABETES

In diabetic patients HA levels correlate with various disease complications. HA serum levels were significantly higher in diabetic patients than in normal subjects and correlated with the levels of fasting plasma glucose and CRP as well as the body mass index (BMI). Diabetic complications like retinopathy and nephropathy were associated with higher HA serum levels (Figure 13). The HA level also correlated with the occurrence of diabetic angiopathy [37, 39]. In over-weight diabetes patients hypertrophic fat cells showed an increased synthesis of hyaluronic acid causing a chronic inflammation of the adipose tissue [16].

3.5 OTHERINDICATIONS

Increased serum levels of hyaluronic acid were observed during different inflammatory processes like psoriasis or sclerosis and also during septic conditions [6]. In sepsis increased levels of hyaluronic acid were found to correlate with disease severity and prognosis with high values of hyaluronic acid associated with a poorer survival rate [3]. Additionally, concentrations of hyaluronic acid have been determined in various other sample types like perfusi-on and lavage fluids. Markedly increased levels of hyaluronic acid were found in broncho-alveolar lavage fluids of patients with pulmonary inflammation due to farmer’s lung, sarcoidosis or adult respiratory distress syndrome [15].

In summary, serum HA acid is useful to estimate the severity and progression rate of degenerative joint diseases as well as to monitor disease progression. Furthermore, serum HA can be a good indicator to judge the success of appropriate therapies especially in patients with rheumatoid arthritis.HA was measured in synovial fluid of dogs with different stages of osteoarthritis. It was concluded that synovial HA concentrations indicate a trend of osteoarthritic disease activity, but is not suitable for staging the disease [73].

Figure 13: Correlation between HA levels and diabetic complications [39].

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Chinese Shar Pei dogs over-express Hyaluronan Synthase 2 (HAS2) to produce ex-cess HA what gives the breed their characteristic wrinkled appearance. Individual Shar-Pei dogs vary in the number of HAS2 mutation copies of this mutation and multiple copies increase the risk for periodic fever in these dogs; most dogs with more than six copies had fever whereas most dogs with less than four copies did not [75]. This association of HAS2 dysregulation and autoinflammation is of wide interest since the genetic cause of periodic fever syndromes in approximately 60% of human cases remains unexplained. Damaged or degraded low molecular weight HA may be a major trigger of the complement system and autoinflam-mation, which opens a new research field in human (and canine) inflammatory disease.

4 THERAPEUTIC&COSMETICAPPLICATIONS OFHYALURONICACID

HA, a natural component of the vitreous humor of the eye, has found many suc-cessful applications in ophthalmic surgery. By intraocular injection the shape of the anterior chamber is maintained. HA solution also serves as a viscosity en-hancing component of eye drops and as an adjuvant to eye tissue repair. Intra- articular injections of HA, so called viscosupplementation, has gained popularity to relieve knee pain in osteoarthritis patients not responding to other treatments. HA is also used in treatment of articular disorders in horses, in particular those in competition or heavy work. It is indicated for carpal and fetlock joint dysfuncti-ons. It is especially used for synovitis associated with equine osteoarthritis. It can

be injected directly into an affected joint, or intravenously for less localized disorders. Intra-articularly administered medicine is fully metabolized in less than a week [76].Its unique viscoelastic and water retaining nature along with its biocompatibility and non-immunogenicity has led to a number of cosmetic applications. In plastic surgery hyaluronic acid is used as injectable soft tissue filler. It is used for lip augmentation, reduction of folds and wrinkles, and removal of scars. In 2003, the FDA approved HA injections for filling soft tissue defects. HA is also a common ingredient in skin-care products. The presence of HA in epithelial tissue has been shown to promote keratinocyte proliferation and increase the presence of retinoic acid, causing skin hydra-tion. HA interaction with CD44 drives collagen synthesis and normal skin function. Some pharmaceutical companies have recently invented anti-wrinkles creams based on HA, whose efficiency has not been proven yet.

5 MEASUREMENTOFHYALURONICACID

5.1 FACTORSAFFECTINGHYALURONICACIDMEASUREMENTSDay-to-day and circadian variations in HA blood levels seem to be small. However, other factors should be taken into consideration to obtain valid and reproducible results [50].

DemographicfactorsSerum hyaluronic acid is influenced by various individual factors including age, sex and ethnicity. The blood level of hyaluronic acid increases with age with higher levels observed in men than in women [32, 35, 46]. Additionally, it seems to be influenced by the individual’s ethnicity, e.g. higher serum concentrations were observed for caucasian Americans compared to African Americans [5]. In articular cartilage the content of hyaluronic acid increases during aging in combination with a decreasing chain length and therefore molecular mass of the hyaluronic acid molecule

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(Figure 14). Shorter hyaluronic acid chains result in smaller proteoglycan aggregates. Since the size of proteoglycan aggregates significantly affects the compressive properties of articular cartilage, it could be assumed that an incre-asing content of hyaluronic acid aims to compensate the decreasing chain length in order to maintain the overall aggregation of proteoglycan monomers and thus to keep the compressive properties of cartilage constant [19, 58].

Foodintake/dietCaloric restriction as well as the dietary composition affected the level of hyaluronic acid in rats (64). In humans, food intake was found to significantly influence the blood level of HA (within 30 minutes) independent from fat content (caloric intake was nearly identical).In patients with chronic liver disease the post-prandial concentrations of hyaluronic acid were found to be signifi-cantly increased compared to healthy subjects and to correlate with disease severity. Patients with liver cirrhosis con-stantly showed values far beyond patients with different stages of liver fibrosis (Figure 15). Analysing post-prandial serum levels of hyaluronic acid in patients with chronic hepatitis might be useful to assess the stage of fibrosis as well as for the diagnosis of liver cirrhosis [21].Since singular post-prandial values might suggest hepatic fibrosis/cirrhosis even in healthy subjects, samples should be taken after overnight fasting to differentiate between healthy individuals and patients with chronic liver disease. A fasting period of at least five hours before sample collection should be adhered [8, 21].

PhysicalactivityPhysical activity elevates the blood level of HA, since muscular exercise improves lymph circulation thus promoting the removal of hyaluronic acid from tissues [18].

Figure 14: Age dependent changes of HA content (left) and molecular mass (right) in articular cartilage [19].

Figure 15: Post-prandial serum levels of HA in healthy subjects (HS), in patients with chronic hepatitis (CH) and with liver cirrhosis (LC) [21].

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ComorbiditiesSince distributed ubiquitously in the organism, HA is not a tissue specific marker. Thus, concentrations of hyaluronic acid determined by blood samples depict the entire body’s HA turnover and coexisting sources of increased HA will contribute to increased blood levels. Consequently, comorbidities increasing concentrations of HA on its own (e.g. diabetes) might shade the disease-specific value of the blood level. In degenerative joint disease, higher levels of hyaluronic acid were observed when more joints were affected [5].

DrugsSubstances impacting liver function can increase the concentration of serum hyaluronic acid. Actively drinking patients with alcoholic liver disease showed significantly decreased levels of serum hyaluronic acid after four weeks of abstinence [31, 60].

SampletypeDue to its metabolization route the concentrations of HA markedly vary between different sample types. The highest concentrations are found in the synovial fluid whereas the concentrations found in urine and blood are significantly smaller (see also Figure 4) [29].

5.2 NORMALVALUESOFHYALURONICACID

ReferencevaluesSerum samples from 53 healthy individuals (16 to 79 years, mean 45.5 years) were tested using the TECOmedical Hy-aluronic Acid Plus Sandwich ELISA.

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TECOmedical 15

HYALURONICACID-HYALURONAN(HA)

Cat. No. TE1017-2 Tests 96 Method ELISA Range 15 – 1000 ng/mL (Pharmacopeia Standard) Min.Det. Conc. 2.7 ng/mL Incubation time 3 hours Sample volume 100 µl (dilute 1:50) Sample type Serum, EDTA plasma and cell supernatant Sample preparation Fasting blood collection. Serum and EDTA plasma stable for 72 hours at 2-8°C, 6 months at -20°C, longer storage at -80°C Maximum 3 freeze- and thaw cycles

Reference values Hyaluronic Acid Values are depending on age and gender and influenced by food intake and physical activity. Clinically Healthy Subjects (n=53) between 16 and 79 years. 36.7 ±23.5 ng/mL.

Mean (ng/mL) SD (ng/mL) Female premenopausal 20.1 14.3

Female postmenopausal 50.3 19.9

Male 42.6 24.6

Cut-off : 90 ng/mL

Species Human

Intended use Diagnosis and monitoring of liver fibrosis and cirrhosis Diagnosis and monitoring of joint diseases HA is also important in diabetes, as a tumor marker and in pulmonary infections.

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