Clinical aspects of monosodium urate monohydrate crystal deposition disease (gout)

Gout is a clinical syndrome with a limited range of manifestations arising as a result of the deposition of crystals of monosodium urate, the final product of purine metabolism in humans. Hyperuricemia is a common chemical aberration that is most often mild and remains asymptomatic. Thus, hyperuricemia should be distinguished from gout, even though urate supersaturation is necessary for the expression of gout. Uric acid overproduction and diminished renal uric acid excretion are the major mechanisms resulting in hyperuricemia, and an understanding of the basis of hyperuricemia in individual gout patients is an important step in determining appropriate treatment and in identifying underlying disorders, offending drugs and toxins, and inherited enzyme defects, all of which can result in hyperuricemia and gout. A scheme is presented for the evaluation of patients with new-onset gout, along with a discussion of the relationships between gout/hyperuricemia and a variety of metabolic disorders that are unusually prevalent in gouty populations.     

Nucleation of monosodium urate crystals.

(1) Calcium greatly increased crystallization of monosodium urate in otherwise pure water, by enhancing both nucleation and growth. (2) Acid accelerated urate nucleation, both by its direct action and indirectly by increasing the free calcium in physiological fluids. (3) Synovial fluid from one gouty patient accelerated urate nucleation, while that from one rheumatoid patient inhibited nucleation. (4) X-rays, collagen, ethyl alcohol, cupric ion, and potassium ion all had negligible influence on urate nucleation. (5) Mechanical shock greatly increased urate nucleation.     

Membranolytic effects of monosodium urate monohydrate: influence of grinding

The effect of grinding on the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and intact erythrocyte membranes was studied. Crystals were ground for between 1-72 h, and percent hemolysis and zeta potentials determined. A cationic amphiphilic probe (CAT12) was incorporated into the erythrocyte membrane and incubated with MSUM. Increasing grinding times caused a decrease in both the crystallinity and zeta potential of the samples, a decrease in percent hemolysis values and a change in the distribution of free and bound spin label populations. The probe redistribution is thought to be due to an electrostatic interaction between negatively charged MSUM and the CAT12 probe.     

Ultrasonography shows disappearance of monosodium urate crystal deposition on hyaline cartilage after sustained normouricemia is achieved

This study aimed at determining whether lowering serum urate (SU) to less than 6 mg/dl in patients with gout affects ultrasonographic findings. Seven joints in five patients with monosodium urate (MSU) crystal proven gout and hyperuricemia were examined over time with serial ultrasonography. Four of the five patients were treated with urate lowering drugs (ULDs) (allopurinol, n = 3; probenecid, n = 1). One patient was treated with colchicine alone. Attention was given to changes in a hyperechoic, irregular coating of the hyaline cartilage in the examined joints (double contour sign or “urate icing”). This coating was considered to represent precipitate of MSU crystals. Index joints included metacarpophalangeal (MCP) joints (n = 2), knee joints (n = 3), and first metatarsophalangeal (MTP) joints (n = 2). The interval between baseline and follow-up images ranged from 7 to 18 months. Serial SU levels were obtained during the follow-up period. During the follow-up period, three patients treated with ULD (allopurinol, n = 2; probenecid, n = 1) achieved a SU level of <6 mg/dl. In two patients, SU levels remained above 6 mg/dl (treated with allopurinol, n = 1; treated with colchicine, n = 1). At baseline, the double contour sign was seen in all patients. In those patients who achieved SU levels of <6 ml/dl, this sign had disappeared at follow-up. Disappearance of the double contour sign was seen in two knee joints, two first MTP joints, and one MCP joint. In contrast, disappearance of the double contour sign was not seen in patients who maintained a SU level ≥7 mg/dl. In one patient treated with allopurinol, SU levels improved from 13 to 7 mg/dl during the follow-up period. Decrease, but not resolution of the hyperechoic coating was seen in this patient. In the patient treated with colchicine alone, SU levels remained >8 mg/dl, and no sonographic change was observed. In our patients, sonographic signs of deposition of MSU crystals on the surface of hyaline cartilage disappeared completely if sustained normouricemia was achieved. This is the first report showing that characteristic sonographic changes are influenced by ULDs once SU levels remain ≤6 mg/dl for 7 months or more. Sonographic changes of gout correlate with SU levels and may be a non-invasive means to track changes in the uric acid pool. Larger prospective studies are needed to further assess these potentially important findings.     

Plasma and synovial fluid as solvents for monosodium urate.

In-vitro differences in monosodium urate (MSU) crystal dissolution in paired plasma and synovial fluid samples from patients with various arthritides were studied. Plasma was a significantly better solvent for MSU than synovial fluid (overall difference 6.3 mg/dl (0.37 mmol/l); significant at P less than 0.001). Attempts to correlate the solubility differentials with the principal compositional differences between the 2 fluids were only partially successful. (1) A tendency towards higher MSU solubility at higher protein levels was observed, but it was too slight to reach statistical significance. (2) Hyaluronidase treatment of synovial fluid significantly enhanced its ability to dissolve MSU (overall difference 2.2 mg/dl (0.13 mmol/l); significant at P less than 0.01) but not sufficiently to explain wholly the plasma-synovial fluid differential.     

Prevention of neutrophil apoptosis by monosodium urate crystals

The effects of monosodium urate (MSU) crystals on apoptosis of cultured peripheral blood neutrophils were investigated. MSU crystals at low concentrations decreased the rate of spontaneous apoptosis of neutrophils in a dose- and time-dependent manner. The culture supernatant of MSU crystal stimulated neutrophils also promoted a delay in neutrophil apoptosis. MSU crystals at higher concentrations rapidly caused cell lysis. These findings indicated that MSU crystals are capable of amplifying the inflammatory responses of gouty arthritis by decreasing the rates of neutrophil apoptosis at lower concentrations and inducing cell lysis at higher concentrations.     

Adsorption of polymorphonuclear leukocyte lysosomal enzymes to monosodium urate crystals

Monosodium urate (MSU) crystals induced prompt release of iysozyme, and slower release of β-glucuronidase, α-mannosidase, and lactic dehydrogenase (LDH) from polymorphonuclear leukocytes. At increasing crystal concentrations, an increasing delay in the apparent onset of β-glucuronidase release was detected which appears to be due to selective adsorption of enzyme activities to the MSU crystals: β-glucuronidase > LDH > α-mannosidase > lysozyme. Lysosomal enzyme adsorption by MSU crystals may then contribute to experimental error or may modulate gouty inflammation.     

Immunoglobulins on the surface of monosodium urate crystals: an immunoelectron microscopic study

The ability of monosodium urate (MSU) crystals to bind immunoglobulins from human serum was investigated by immunoelectron microscopy. Synthetic MSU crystals were incubated with serum and then processed with an indirect immunoperoxidase technique which used primary antibodies directed against human IgG, IgA or IgM, respectively specific for Fc fragments, alpha chains, and mu chains. Results were analyzed by transmission electron microscopy on intact crystals dried on formvar coated grids, and in thin sections. Both techniques demonstrated the binding of IgG, IgA and IgM to MSU crystals and the availability of the Fc fragment of the crystal bound IgG for immunologic reactions.     

Mechanisms of cellular interaction with monosodium urate crystals. igg-dependent and igg-independent platelet stimulation by urate crystals

Monosodium urate crystals (MSU) stimulate suspensions of washed platelets or neutrophils. When MSU crystals are coated with IgG, as occurs in plasma, stimulation is markedly enhanced. These studies which use MSU-induced human platelet serotonin secretion as a model examine the nature of cellular recognition mechanisms for MSU crystals and IgG-coated MSU crystals. F(ab')₂ fragments of specific anti-Fc antibody blocked and the lipopolysaccharide of Salmonella minnesota R595 enhanced human platelet secretion induced by IgG–coated urate crystals. These agents had little effect on stimulation by uncoated crystals. This indicated that urate crystals stimulate platelets independently of fluid phase IgG. Urate crystals directly stimulated suspensions of washed rabbit platelets which lack Fc receptors. In contrast to human cells, stimulation was blocked by IgG. This again demonstrated IgG-independent cell stimulation by urate crystals. Calcium pyrophosphate dihydrate crystals could trigger human platelet secretion only when coated with IgG. This suggests that when crystals are coated with IgG, the surface-bound IgG alone may be the stimulus to the cell. This was supported by the finding that polyvinylpyridine-N-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated, but not IgG-coated, urate crystals. These data indicate that urate crystals (and potentially other surface or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of adsorbed IgG or, when coated with IgG, by triggering the cell via immunoglobulin receptors.     

Polymorphonuclear leukocyte responses to monosodium urate crystals: modification by adsorbed serum proteins

The effects of protein absorption to monosodium urate monohydrate (MSU) crystals on crystal-induced lysosomal enzyme release from human neutrophils were studied. Native crystals produced prompt release of both lysosomal and cytoplasmic enzymes, suggesting that they are directly membranolytic for the neutrophil. When albumin, IgM, or beta-lactoglobulin were adsorbed to MSU crystals, lysis was blocked and lysosomal enzyme release was negligible. Prior incubation of crystals with 20% human serum or adsorption of IgG resulted in inhibition of lysis but enhancement of lysosomal enzyme liberation. This non-cytolytic lysosomal enzyme release induced by IgG-coated crystals may be important in the pathogenesis of acute gouty arthritis.     

Binding of synovial fluid proteins to monosodium urate crystals in vitro

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blot techniques were used to analyze proteins adsorbed in vitro to synthetic monosodium urate crystals (MSUC) from a variety of gouty biologic fluids. Distinct differences in adsorbed proteins were found in comparing synovial fluid (SF) with serum or plasma, particularly at 220 kD and below 19 kD. Further studies should consider the importance of the synovial milieu. Patterns of proteins adsorbed to MSUC from the SF of all 12 patients with gout were similar. A considerable number of polypeptides appeared to be selectively adsorbed. Of these, polypeptides associated with fibronectin, C1q and IgM were identified by immunotransblotting. The experiments also demonstrated that the overall polypeptide patterns obtained by SDS PAGE of eluates from MSUC exposed to SF were unaffected by heating MSUC to 180 degrees C, as well as by the volume of SF in the incubation mixture.     

A comparison of five preparations of synthetic monosodium urate monohydrate crystals.

We conducted preliminary crystallographic investigations and biologic studies on 5 synthetically grown preparations of monosodium urate monohydrate (MSUM) crystals including one preparation of urate spherulites. The 5 crystal preparations exhibited wide variations in morphology, size, surface area, and ultrastructure, but only a few changes in their biologic effects. When injected intraarticularly, urate spherulites were less phlogistic than most acicular forms. Since crystal-cell interactions and inflammatory responses are influenced by crystal size, morphology, and surface characteristics, standardization of methods of preparing MSUM crystals is, therefore, important, particularly when analyzing and comparing results from different research laboratories.     

The added value of synovial fluid centrifugation for monosodium urate and calcium pyrophosphate crystal detection

The aim of the study was to assess the added value of synovial fluid (SF) centrifugation for microscopic monosodium urate (MSU) and calcium pyrophosphate (CPP) crystal detection in patients with arthritis. This is a prospective observational study using SF samples from joints of patients undergoing joint arthrocentesis. Two blinded observers assessed the SF smears by polarized light microscopy for the presence of crystals before as well as after centrifugation. SF samples were collected from 98 patients with arthritis. After exclusion, 87 samples were eligible for inclusion. Of each sample, 2 smears before and after centrifugation were prepared and microscopically examined, resulting in 348 smears per observer. Observer 1 identified MSU crystals in 18.4% and CPP in 9.2% of the smears before as well as after centrifugation. No extra MSU crystal-positive smears were identified after centrifugation. However, centrifugation yielded 4 additional CPP crystal-positive smears. Observer 2 identified MSU crystals in 15.5% and CPP crystals in 6.3% of the smears before as well as after centrifugation. Centrifugation yielded 2 additional MSU crystal-positive smears and 4 CPP crystal-positive smears. Monosodium urate crystals were well recognized without centrifugation. Centrifugation of SF had limited additional value for increasing the amount of MSU-positive smears. However, CPP crystals were identified in a higher number of smears after centrifugation than before. Therefore, centrifugation may be of additional value in selected patients with suspected calcium pyrophosphate deposition disease and to a lesser extent for gout.     

A spin label study of the membranolytic effects of crystalline monosodium urate monohydrate

The nature of the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and phospholipid membranes was studied using electron spin resonance. Two spin probe molecules were incorporated into intact human erythrocytes and incubated with MSUM crystals. The apparent increased fluidity of 5-doxyl stearic acid incorporated erythrocytes after a 2 h incubation with MSUM was probably due to an electrostatically induced redistribution of probe from the outer more rigid layer to the fluid inner leaflet via a flip-flop mechanism. It was suggested that the MSUM induced redistribution of cationic amphiphilic probe population in the whole erythrocyte was also due to an electrostatic interaction between negatively charged MSUM crystals and positively charged probe. Possible mechanisms of MSUM induced membranolysis are discussed.