Alfred Jay Bollet

Biochemical Findings in Normal and Osteoarthritic Articular Cartilage. II. Chondroitin Sulfate Concentration and Chain Length, Water, and Ash Content*

Softening and erosion of cartilage, the characteristic early lesions of osteoarthritis, occur in sites of decreased chondroitin sulfate concentration. The role of aging and the specific biochemical events that produce this alteration in glycosaminoglycan composition have been under study in several laboratories. Collins and McElligott found increased radiosulfate fixation in these lesions compared with normal articular cartilage from the same individuals, suggesting increased synthesis of polysaccharide (1). These observations indicated an increased turnover of chondroitin sulfate in osteoarthritic cartilage lesions, and attention has become focused on the mechanism responsible for breakdown of this glycosaminoglycan. As reported previously (2), when the cartilage proteinpolysaccharide complex was extracted from human articular cartilage obtained at autopsy, a decrease in the concentration of chondroitin sulfate but not protein was found in the areas of erosion. This observation pointed to a specific breakdown of the glycosaminoglycan, since proteolysis might be expected to cause removal of both components such as occurs after papain injection into rabbits (3). An enzyme resembling testicular hyaluronidase could be responsible, but reports of the presence of hyaluronidase in tissues other than testis were unconvincing or unconfirmed. A reevaluation of the tissue distribution of this enzyme resulted in the finding of an enzyme with most of the characteristics of testicular hyaluronidase in

Acid Mucopolysaccharides in Normal Serum

Mucopolysaccharide which contains uronic acid has recently been described in normal plasma (1, 2). A method for the quantitation of plasma acid mucopolysaccharide was described by Badin, Schubert and Vouras (1), based on the observation that chondroitin sulfate added to plasma is found in euglobulin precipitates (3). These authors reported an increased amount of acid mucopolysaccharide in the plasma of 10 patients with rheumatoid arthritis (1). The study of acid mucopolysaccharides in blood which are chemically related to those of connective tissue might reveal information of interest concerning the diseases which primarily affect connective tissue. Accordingly, a relatively practical method was devised for measurement of the uronic acid-containing polysaccharide in the presence of large amounts of protein, and studies of the nature of the mucopolysaccharides present in blood were undertaken. Observations made on plasma and serum from normal adults are reported.

Edema of the Bone Marrow Can Cause Pain in Osteoarthritis and Other Diseases of Bone and Joints

Cartilage degeneration, although fundamental to the pathogenesis of osteoarthritis, is not the site of origin of pain, the predominant symptom of osteoarthritis. Peripheral nerves generally follow the path of blood vessels, and cartilage contains no nerves or blood vessels. Pain can originate in the synovium, the joint capsule, or intra-articular ligaments, as well as in the muscles and soft tissues, which surround and move the joint in osteoarthritis. Ordinary radiographs show the effect of degeneration of joint cartilage as narrowing of the space between the surfaces of the articular bone (usually called the joint space or, more accurately, the cartilage space). However, patients can have considerable pain despite a normal-looking cartilage space, or pain can be mild despite marked narrowing. Likewise, other radiographic changes seen in osteoarthritis, including thickening of the subchondral bone (eburnation) and formation of osteophytes, do not correlate with the presence or the severity of pain. The clinical significance of joint pain can therefore be obscure and difficult to determine. In this issue, Felson and colleagues (1) describe edema in the subarticular bone marrow adjacent to the knee, detected by T2-weighted magnetic resonance imaging (MRI), in patients with painful osteoarthritis of that joint. Edema of the bone marrow has also been demonstrated in patients with traumatic bone injuries, including bone bruises and other forms of overuse (2); the pathologic process in the subarticular marrow of patients with osteoarthritis is probably an analogous phenomenon. Thinning and erosion of the cartilage in osteoarthritis decrease the protection of the underlying bone afforded by the articular cartilage, which loses its capacity to absorb impact stresses and minimize friction during joint motion. The increase in physical stresses transmitted to the subchondral bone results in cortical thickening; the increased density of the bone further decreases the dampening of physical stresses, especially impact stresses, which are thus transmitted more fully to the underlying trabecular bone and bone marrow. Edema of the bone marrow seen on MRI reflects an inflammatory response to these traumatic forces. Afferent nociceptive nerve fibers containing the neurotransmitter substance P are found in periarticular tissues, including the periosteum and subchondral bone, of patients with osteoarthritis (3). Pain can arise from pathologic processes in these structures. Patients with osteoarthritis of the knee often report no or minimal pain while walking but considerable pain after activity, especially at night. These delayed responses can be explained by findings such as those reported by Felson and colleagues (1); they reflect the time it takes for the marrow spaces (visualized by using MRI) to react. Edema of the bone marrow has also been observed in patients with painful, transient osteoporosis (4, 5), and periosteal edema along with marrow edema has been seen on MRI in patients with otherwise unexplained medial tibial pain after trauma (6). Magnetic resonance imaging has demonstrated other bone marrow lesions in patients with bone pain, such as those with sickle-cell crises (7). Magnetic resonance imaging can also detect early subarticular erosions in rheumatoid arthritis (8). Impaired venous drainage from the bone marrow has been suggested as a cause of pain in patients with osteoarthritis, since the resulting venous hypertension would increase intraosseous pressure in the closed spaces of the bone marrow compartments (9). Such venous hypertension would contribute to the development of marrow edema and may be an aspect of the phenomenon that Felson and colleagues observed (1). The development of venous hypertension and bone marrow edema may also be related to the development of cysts in the subchondral bone in osteoarthritis. Edematous changes in the bone marrow early in the evolution of avascular necrosis of the femoral head (10) and in premonitory lesions of osteonecrosis in subchondral areas of the tibia have been reported in patients with osteoarthritis (1). These observations explain pain that occurs before changes indicative of osteonecrosis are visible on standard radiologic techniques, and they emphasize the potential value of therapeutic intervention before positive radiographs are obtained. Core decompression of the femoral head has been recommended to minimize the effects of increased pressure early in the development of avascular necrosis (12). Scintigraphy has revealed changes observed in the subchondral bone during the development of osteoarthritis. These changes reflect the increased blood flow and formation of bone that occur in response to increased physical stresses resulting from pathologic processes in the cartilage (13). Such findings have been reported to indicate a high likelihood of progression of osteoarthritis, and some have suggested that new therapeutic measures directed toward them are needed (14). Scintigraphic studies are also positive early in the development of osteophytes. Although the presence of fully developed osteophytes on radiography does not correlate with the occurrence of pain in patients with osteoarthritis, these lesions can be painful early in their development. Such early lesions are easily visible in the distal interphalangeal joints, where a developing Heberden node can be painful, warm, red, swollen, and tender. Ordinary radiographs would be negative at this point, except for swelling of the soft tissue. Later, when the lesion is fully developed, the gnarled finger is no longer painful, tender, or warm, but the radiograph shows osteophytosis. The same phenomena that occur in the evolution of these visible lesions presumably occur in sites deep in muscle and soft tissue and constitute another reason for the lack of correlation of symptoms with ordinary findings on radiography in patients with osteoarthritis. Felson and colleagues (1) show a fair correlation between occurrence of pain and presence of bone marrow edema, especially when cartilage loss is severe, as judged by extreme narrowing of the cartilage space on standard radiographs. Although not all osteoarthritis is exactly the same disease, these observations elucidate the pathologic processes in the underlying bone and bone marrow in osteoarthritis of the knee and presumably apply to other joints afflicted with the same type of disease process. No correlation was observed between bone marrow edema and severity of pain as measured by the standard scales used in clinical studies of osteoarthritis. The greater the degree of cartilage erosion seen on standard weight-bearing radiographs, however, the higher the frequency of observable bone marrow lesions; all patients with grade 4 osteoarthritis (complete loss of the cartilage space) had severe edema. A close correlation between the presence of edema and the presence of pain cannot be expected in a clinical phenomenon that can arise from so many different structures, as is the case with pain in osteoarthritis. Longitudinal studies, in which the presence of pain can be correlated with the appearance and disappearance of bone marrow edema, would more definitively establish that marrow edema is a cause of pain; however, as Felson and colleagues point out, such studies would be far too expensive at this time. In view of the observation that bone marrow edema is found in most patients with painful osteoarthritis of the knee, as well as in other syndromes associated with juxta-articular pain, such as bone bruises and osteonecrosis, we can conclude that edematous changes in the subcortical bone marrow are one of the many sources of pain in patients with osteoarthritis. At present, this observation helps more in considering measures to minimize the stimuli that produce the pain rather than in treating pain once it develops. Improved understanding of the pathogenesis of symptoms of osteoarthritis can lead to improved treatment.

The Measurement of Tissue Acid Mucopolysaccharides

Investigation of the metabolism of connective tissue acid mucopolysaccharides in normal and abnormal states has been hampered by a lack of practical analytical methods. Reported methods for measuring tissue acid mucopolysaccharides involve multiple steps of enzyme digestion and precipitation (1, 2), with consequent risk of loss of material as well as poor adaptability for multiple analyses. This report describes a less complex procedure which gives reproducible results and good recovery of test material. Values are given for the acid mucopolysaccharide content of human dermis, subcutaneous tissue, aorta and thyroid gland, using both orcinol and carbazole reactions for uronic acid. Chromatographic and electrophoretic studies of the polysaccharides found and determination of the proportion digested by testicular hyaluronidase are also reported.

MUCOPOLYSACCHARIDE CONTENT OF SKIN IN PATIENTS WITH PRETIBIAL MYXEDEMA

It is well-known that the skin of patients with primary and secondary myxedema contains a mucinous infiltrate. This extracellular deposition of metachromatically staining material has been interpreted as being a complex of acid mucopolysaccharide and protein (1). It has not been defined chemically. The infiltrate disappears from the skin of patients with generalized myxedema who are given 120 to 180 mg. of desiccated thyroid daily for six to eight weeks (1). A deposit, histochemically like that which occurs in generalized myxedema, occasionally presents locally in the pretibial skin of patients with thyrotoxicosis and exophthalmos (2, 3). Whenthe patient with localized pretibial myxedema is given 180 mg. of desiccated thyroid a day, however, no clinical or histochemical change occurs in the myxedematous plaque. In 1947 Watson and Pearce isolated a crude mucopolysaccharide fraction from the pretibial plaques of two patients with pretibial myxedema. They found considerably less of this material in skin from the amputated leg of an elderly man (4). Wehave found no record of the comparison of chemical or histochemical findings of myxedematous areas to other areas of the skin of patients with pretibial myxedema, or comparison with control patients of similar age. Wewish to report a chemical study of the mucopolysaccharide concentration of the skin of six euthyroid patients with pretibial myxedema and exophthalmos and eight patients of comparable

Metabolism of mucopolysaccharides in connective tissue. I. Studies of enzymes involved in glucuronide metabolism.

Details of the intermediary metabolism of mucopolysaccharides have been studied in microorganisms and some animal tissues (1). Wehave undertaken an exploration of enzymatic steps in the synthesis of mucopolysaccharides in extracts of the connective tissue which enter polyvinyl sponges inserted under the skin of guinea pigs and extracts of human synovial tissue obtained surgically. Uridine diphosphoglucuronic acid (UDPGA) is presumed to be the donor of glucuronic acid in the synthesis of acid mucopolysaccharides (1). This report concerns primarily the enzymatic steps involved in the synthesis of UDPGAfrom glucose, and other enzymes involved in the metabolism of glucuronides, including glucuronosyl transferase and beta-glucuronidase. Schematic outline of the actions of the enzymes studied is given in Table I.

Effect of acute clinical stress on the levels of hexosamine in serum and its excretion in urine.

The total serum hexosamine 2 level is elevated in many unrelated chronic diseases (1, 2), and in experimental animals following acute injury (3, 4). It is not clear whether this elevation is due to increased production, diminished utilization or merely a shift of tissue hexosamines into the circulation. Since the activity of the adrenal glands is increased in many of these conditions of acute and chronic stress, the role of the adrenal cortex iti sontrclling serum hexosamine levels has been studied. During the course of these studies, it was noted that normal urine contained relatively large amounts of hexosamine, which also increased during conditions of stress. The excretion of hexosamine in the urine in a variety of clinical situations has been measured, therefore, for the purpose of defining factors which alter the rate of its excretion and of correlating excretion levels with changes in serum hexosamine. An ion exchange technique has been applied to urine hydrolysates for the purpose of separating hexosamines from the many chromogens in urine which interfere with the colorimetric determination of hexosamine (5). Previously reported studies on urinary hexosamine have not taken this into account and consequently report erroneously high values (6, 7).

Observations on the use of prednisone in patients with progressive systemic sclerosis (diffuse scleroderma).

Excerpt Both cortisone and corticotropin have been reported as capable of inducing favorable changes in the cutaneous and visceral symptoms of patients with progressive systemic sclerosis.1-6While ...

EFFECTS OF ANTIBODIES ON MITOCHONDRIA

Since the demonstration of the lupus erythematosus phenomenon by Hargraves, Richmond, and Nforton (1), a great deal of attention has been paid to the numerous antibody-like serum factors in patients with active systemic lupus erythematosus (SLE). Until recently, this attention has been concentrated on those factors that react with whole nuclei or nuclear subunits, such as DNAand histone. Asherson (2) and Deicher, Holman, and Kunkel (3) now have demonstrated other common serum factors in SLE that fix complement with cytoplasmic components, including mitochondria and microsomes from a variety of animal and human sources. The observation of low serum complement levels in active SLE (4-10) suggests the possibility that complement-fixing antigen-antibody interactions might occur in vivo and play a role in the pathogenesis of the disease. Antibodies which react with these mitochondria in. vivo might interfere with important metabolic functions, since mnitochondria contain enzymes involved in the regulation of oxidation and highenergy bond formation. A study of the effect of induced antimitochondrial antibodies in rabbits on mitochondrial enzyme activity was therefore undertaken to define the possible metabolic effects of such antibodies preliminary to study of the effects of the antimitochondrial antibodies found in SLE. This report describes several effects of antimitochondrial serum in various immunologic systems; such serum caused inhibition of mitochondrial enzymes, particularly those in the electron transport chain. A heat-labile serum component, presumably complement, potentiated inhibition of DPNHoxidase by mitochondrial antibody.

Systemic lupus erythematosus.

Lupus is a serious, potentially life-threatening disease. Its natural history is to culminate in a crescendo of autoimmunity and organ involvement. Difficulty arriving at the diagnosis may delay institution of therapy, which although helpful brings new problems. The fundamental understanding of this disease has made major strides. There is no consensus on the likely etiological agent(s), though evidence implicates Epstein-Barr virus, and the genetic approach to understanding etiology is in its infancy. Nevertheless, efficacious treatments are anticipated in the plethora of new biologics likely to have therapeutic effects in lupus, essentially a disease of immune dysregulation.This document relates to methods and materials involved in diagnosing SLE. For example, this document provides arrays for detecting polypeptides that can be used to diagnose SLE in a mammal. In addition, methods and materials for assessing SLE activity, determining the likelihood of experiencing active SLE, and detecting SLE treatment effectiveness are provided herein.