Conversion of superoxide generated by polymorphonuclear leukocytes to hydroxyl radical: a direct spectrophotometric detection system based on degradation of deoxyribose.

Hydroxyl radical is produced secondarily after phagocytic cells have been stimulated to generate superoxide anion. The systems used most commonly for detection of cell-generated hydroxyl radical are often inconvenient for routine biomedical research. We have modified an assay used heretofore in cell-free systems, that is, the degradation of deoxyribose, and adapted it for use with neutrophils. The time and dose responses of the system, requirement for chelated iron, inhibition profiles with various scavengers, and correlation with superoxide production have been ascertained. The method correlated strongly with a standard but more cumbersome technique. Values for a normal population are provided. The method can readily be used to study the parameters of superoxide-hydroxyl radical conversion by cells in various disease or treatment states.

In vitro sensitivity of the three mammalian collagenases to tetracycline inhibition: relationship to bone and cartilage degradation.

There are at least nine tetracycline (TC) analogs (both antimicrobial and nonantimicrobial) with documented capacity to inhibit, both in vitro and in vivo, the connective tissue degrading activity of matrix metalloproteinases (MMPs). Of the three MMPs that can degrade native helical collagens, MMP-13 (initially identified as rat osteoblast and human breast cancer collagenase, and now known to also be expressed by human cartilage and bone cells) is the most sensitive to TC inhibition (IC50 values in vitro generally less than 1 microgram/mL); the TCs inhibit both the collagenolytic as well as the gelatinolytic activity of this enzyme. The IC50 for MMP-8 (neutrophil collagenase) in vitro ranges from 15 to 86 micrograms/mL depending on assay conditions and choice of TC, whereas inhibition of the fibroblast enzyme (MMP-1) generally requires levels in excess of 200 micrograms/mL (except for CMT-3). The TC compounds that are highly effective against MMP-13 in vitro are also highly inhibitory of glycosaminoglycan release from interleukin-1-stimulated cartilage explants in culture. The current data correlate well with: (i) literature values for TC inhibition of bone resorption by isolated osteoclasts; (ii) inhibition by TCs of avian tibial resorption in organ culture; and (iii) the dramatic ability of TCs to inhibit bone destruction in many rat models (rats have only MMP-8 and MMP-13, and no MMP-1). By carefully selecting a TC-based MMP inhibitor and controlling dosages, it should be possible to inhibit pathologically excessive MMP-8 and/or MMP-13 activity, especially that causing bone erosion, without affecting the constitutive levels of MMP-1 needed for tissue remodeling and normal host function; in this regard, three newly developed CMTs (especially CMT-8, and, to a lesser extent, CMT-3 and -7) appear to be most effective.

Glycerol-induced hypoglycemia: a syndrome associated with multiple liver enzyme deficiencies. Clinical and in vitro studies.

A 4 10/12 yr-old white male presented with a history of occasional grand mal seizures and hypoglycemic episodes after overnight fasting. Upon evaluation, he became hypoglycemic after 1 g/kg oral glycerol challenge (plasma glucose: 31 mg/dl in 45 min), but had normal glucose, alanine and fructose tolerance tests. He responded well to a glucagon challenge after 11 hr fast but he became hypoglycemic and could not normalize his blood glucose after a 2nd glucagon stimulation test after 17 hr of fasting. Studies conducted on a percutaneous liver biopsy, and compared with 3 non-hypoglycemic controls, showed reduced activities (20%-30% of normal) of alpha-glycerophosphate dehydrogenase, alpha-glycerophosphate oxidase and fructose-1,6-diphosphatase. Alpha glycerophosphate in the patient's liver was elevated. Two types of electrophoresis showed absence of one enzymatically active zone and overall decrease of staining intensity for alpha-glycerophosphate dehydrogenase. Other liver enzymes tested were normal. The 50% inhibition of the patient's liver fructose-1,6-diphosphatase by alpha-glycerophosphate occurred, in vitro, or lower concentration than in controls (11 versus 22-40 mM). Electron microscopy revealed hepatocytes with moderately swollen mitochondria that very occasionally contained dense inclusions in the inner mitochondrial matrix. After discharge from the hospital, the patient followed a normal course, with a regimen of multiple snacks and avoidance of high-fat food in the morning.

Inhibition of epiphyseal cartilage collagenase by tetracyclines in low phosphate rickets in rats.

Drugs in the tetracycline family can inhibit mammalian tissue collagenase both in vitro and in vivo by a mechanism that is independent of antibiotic action. The epiphyseal cartilages of rachitic rats contain extremely high levels of collagenase (CGase), and we have used this model to study further the phenomenon of tetracycline inhibition of tissue CGase. Rickets was induced in rats by phosphate/vitamin D deficiency and parameters of gross bone morphology, bone chemistry, and serum chemistry were evaluated in both rachitic and nonrachitic animals with and without treatment with oral tetracyclines (TETs). Minocycline (or doxycycline) partially suppressed the appearance of many of the expected changes in the rachitic animals, including gross bone hardness, growth plate widening, long bone length, suppression of weight gain, and decreased bone ash content. The effects were dose dependent and were associated with marked suppression of the enhanced CGase activity. Examination of collagen breakdown products by SDS-PAGE documented that the rachitic enzyme behaved like other mammalian collagenases including in vitro inhibition with minocycline 10-20 micrograms/ml and with a nonantibiotic tetracycline. No evidence of TET osseous toxicity was noted, and, in fact, administration of TET to nonrachitic animals had a mildly favorable effect on growth and development. TET suppression of CGase can be demonstrated in a well defined model system and this form of pharmacologic enzyme inhibition can be a useful probe for delineating the role of the enzyme in connective tissue pathology.

Placental permeability and energy metabolism enzymes in fetuses of lipemic rats.

A model of maternal lipemia without hyperglycemia, in the rat, produced by high-fat feedings, was developed to study the effects of and abnormal maternal lipid homeostasis on placental transport of nutrients and possible alterations of key enzymes of energy metabolism in the liver and brain of the fetuses. Pregnant rats fed lower concentrations of fat served as controls. All studies were carried out in dams and fetuses one day prior to delivery. The dietary treatment of the dams and fetuses produced in the fetuses ketonemia as well as lipemia. Following a bolus of 14C-3-0-methyl-D-glucose to the dams, the levels of the tracer remained higher in the blood and brain of lipemic than in control fetuses. By contrast, there was a decrease in the fluxes of 14C-alpha-amino-isobutyric acid in the fetuses of lipemic dams as compared to controls. Among enzymes of energy metabolism, fetal liver glucose-6-phosphatase and succinic dehydrogenase were enhanced by lipemia. Fetal brain glucose-6-phosphatase was depressed. Thus, lipemia, as occurring in poorly controlled maternal diabetes, may be a factor in determining the access to the fetus of essential, neutral amino acids and alter the normal activity of energy metabolism enzymes in the fetus.

Effects of lead on gamma-glutamyl transpeptidase and (Na+ -K+)-adenosine triphosphatase of the small intestinal mucosa.

Oral lead, when fed to young rats for 6 weeks, at a level of 0.50 in the diet, produced an increase in small intestinal mucosal gamma-glutamyl transpeptidase, an enzyme capable of catalyzing membrane translocation of amino acids and small peptides. However, a non-competitive inhibition with the glutamate acceptor used, glycylglycine, was demonstrated for lead, in vitro (l50 = 1.0 mM in purified brush border preparations). Lead ingestion caused a reduction of small intestinal mucosal (Na+ -K+)-ATPase (l50 = 0.4 - 0.6mM). At 0.2 mM concentration, lead was a competitive inhibitor for ATP in a (Na+ -K+)-ATPase assay system in vitro. A higher concentration of lead (0.5 mM) also produced an inhibitory, but non-competitive effect, with a decline of Vmax from 36.6 to 8.1 nmoles/min X cm.

Degradation of hyaluronic acid by polymorphonuclear leukocytes.

Degradation (depolymerization) of hyaluronic acid is readily accomplished by superoxide-ion-generating systems, especially those which beget secondary free radicals. It has been presumed, but not confirmed, that this is the mechanism by which neutrophils might alter synovial fluid viscosity. We have demonstrated, in a neutrophil (PMN) superoxide system, physical disruption of the hyaluronate macromolecule using column chromatography and by measurement of intrinsic viscosity. In addition, comparison of calibrated free radical fluxes between a cell-free superoxide system and a neutrophil system revealed very close parallels in iron requirement, inhibition by free radical scavengers, and magnitude of effect. It is concluded that oxygen-derived free radicals are probably the major, if not sole, mechanism by which neutrophils might degrade hyaluronate.

Enhancement of rat serum ceruloplasmin levels by exposure to hyperoxia.

Exposure of rats to elevated oxygen tensions is a well-known method of producing enhanced levels of pulmonary antioxidant enzymes. Ceruloplasmin is a serum constituent which possesses scavenging activity toward oxygen radicals. Rats exposed either to continuous 85% oxygen for 7 days or to intermittent hyperbaric oxygen for up to 19 days developed not only enhanced lung antioxidant enzymes but also increased levels of serum ceruloplasmin. The latter did not appear to be merely an "acute phase reactant" as there was no change in total serum protein, plasma fibrinogen, serum -SH groups, sedimentation rate, or serum iron. Induction of ceruloplasmin may account for some of the anti-inflammatory activities of elevated oxygen tensions.

Degradation of hyaluronic acid by neutrophil derived oxygen radicals is stimulus dependent.

A disparity has been noted between the flux of superoxide generated by neutrophils and the effects of those radicals on a typical macromolecular substrate, hyaluronic acid (HA). To further investigate this phenomenon, comparative degradation studies were conducted using phorbol myristate acetate (PMA), which caused neutrophils to readily degrade HA, and digitonin, a stimulus which, like PMA, produced a sustained superoxide flux but which did not affect HA. The differential effects of the 2 stimuli could be related to concomitant release of peroxidase activity. The implications of this mechanism for in vivo oxygen radical effects are discussed.

The effect of oxygen adaptation on oxyradical injury to pulmonary endothelium.

Rats exposed to 85% O2 for 7 days develop an increase in pulmonary tissue levels of antioxidant enzymes and are able to survive subsequent exposure to 100% O2. In order to examine endothelial cell function in O2-adapted rats, we chemically generated oxygen radicals within the pulmonary circulation of lungs isolated from 85% O2-exposed rats and used [14C]5-hydroxytryptamine (5-HT) uptake as an indicator of endothelial cell function. The [14C]5-HT uptake in lungs of O2-exposed rats was similar to that in air-exposed rats and was reduced by cyanide, anoxia, and by imipramine. After generation of O2-derived free radicals within the pulmonary circulation, there was a progressive decline in [14C]5-HT uptake in both groups, with a more rapid decline seen in the lungs of air-exposed rats. These data suggest that pulmonary endothelial cells that survive 85% O2 exposure for 7 days are capable of normal function and are more resistant to further oxyradical injury.

Inhibition of the proteolytic contaminant in commercial xanthine oxidase preparations by serum protein fractions.

Commercial xanthine oxidase, widely used for generation of oxygen radicals in vitro, is usually contaminated by proteolytic activity, which limits its utility in studies of oxygen radical damage to protease sensitive substrates. An easily prepared fraction of fetal calf serum was found to inhibit virtually all of the proteolytic contaminant without affecting superoxide generation. The effects attainable with the "purified" enzyme were demonstrated with two protease sensitive targets: proteoglycan subunit from cartilage and fibronectin from human plasma.

Protein and lysozyme content of adult human nucleus pulposus.

A radiologically normal human nucleus pulposus was extracted with 4 M guanidinium chloride and the non-collagenous proteins separated from the proteoglycans by dissociative density gradient centrifugation. Lysozyme was identified as a matrix constituent of the normal, mature human nucleus pulposus.

Tetracyclines suppress matrix metalloproteinase activity in adjuvant arthritis and in combination with flurbiprofen, ameliorate bone damage.

Tetracyclines are potent inhibitors of 2 major matrix metalloproteinases which have been implicated in connective tissue degradation: collagenase and Type IV collagenase/gelatinase. We directly identified these enzyme activities in extracts of inflamed paw tissue from rats with adjuvant arthritis. Oral tetracycline therapy suppressed metalloproteinase activity in arthritic tissue, but even very high doses failed to exhibit substantial antiinflammatory efficacy (reduced joint swelling and paw diameter). Flurbiprofen, a conventional nonsteroidal antiinflammatory drug, reduced inflammatory indices as expected. The combination of the 2 agents administered orally completely inhibited collagenase activity, significantly inhibited gelatinase activity and produced substantial normalization of radiographic joint damage, far greater than either drug alone. Tetracycline inhibition curves in vitro suggest that the collagenase in this tissue is not of fibroblast origin. Tetracycline derivatives might be useful adjuncts to prevention of tissue damage in chronic inflammatory arthritides.

Alterations of intestinal and renal functions in rats after intraperitoneal injections of lead acetate.

When lead acetate was administered intraperitoneally to young rats at a dose of 20 mg/kg (five times a week for 6 weeks), their growth rate was retarded when compared with controls injected with sodium acetate. Only a small amount of the heavy metal reached the circulation and exerted limited effects on typical target organs. However, large, electron-dense inclusion bodies were found in the abdominal cavity. The in vivo intestinal absorption of glucose was reduced. When perfused at 40 mM concentration, the experimental animals had a mean absorption rate of 152.1 nmol/min . cm vs. 230.6 in the controls (p less than 0.01). Also, sodium and potassium transport was reduced. No effects were observed on amino acid transport and (Na+-K+)-ATPase. Mg++-ATPase, glucose-6-phosphatase, fructose-1, 6-diphosphatase, pyruvate kinase, succinic dehydrogenase, and tryptophan hydroxylase in the small intestinal mucosa and the kidney were unaltered. Renal alkaline phosphatase was decreased. These studies confirm the greater susceptibility of some active transport mechanisms of the small intestinal mucosa to lead toxicity, compared to those of the kidney.

Effect of tetracyclines which have metalloproteinase inhibitory capacity on basal and heparin-stimulated bone resorption by chick osteoclasts.

Several tetracyclines (TETs) are potent inhibitors of collagenase (CGase) and can inhibit connective tissue degradation in a variety of inflammatory and degenerative disorders. The role of CGase in bone resorption by osteoclasts (OC) remains unclear. Disaggregated OCs from chick embryos were cultured for 24 h on devitalized bovine cortical bone +/- heparin in the presence of various TETs. Doxycycline (Dox) inhibited pit formation in a dose-dependent manner. CMT, a TET derivative which inhibits matrix metalloproteinases (MMPs) but is not antimicrobial, also inhibited chick OC bone resorption. Heparin markedly stimulated bone resorption at 5 micrograms/ml, which was reversed by Dox, 5 micrograms/ml. TETs can reversibly inhibit both basal and heparin-stimulated bone resorption by chick OCs. These findings suggest that MMPs may play a role in osteoclastic bone resorption, and that safe and effective inhibitors of MMPs, including certain TETs, might have a potential therapeutic role.

Administration of systemic matrix metalloproteinase inhibitors maintains bone mechanical integrity in adjuvant arthritis.

OBJECTIVE: To evaluate the influence of systemic tetracycline derived antimetalloproteinase compounds on bone morphology and mechanical integrity. METHODS: Male Lewis rats (n = 78) were randomly assigned to one of 10 groups, comprising controls, adjuvant arthritis (AA), and adjuvant arthritis with various combinations of 2 chemically modified, non-antimicrobial tetracycline derivatives (CMT3 or CMT8) with either of 2 nonsteroidal antiinflammatory agents (flurbiprofen or tenidap). After AA induction (23 days), pharmacological efficacy was assessed by inflammatory indices, body mass changes, joint radiological destruction scores, and pyridinoline collagen derived crosslinks. The structural and material properties of the rat femoral neck were assessed biomechanically. RESULTS: Neither CMT had an antiinflammatory effect, but flurbiprofen and tenidap (alone or together with either CMT) significantly reduced joint inflammation. Pyridinoline excretion increased markedly in untreated AA, but was substantially normalized by either CMT3 alone or by CMT8 with flurbiprofen. AA produced significant deleterious effects on femoral neck structure and mechanical properties. Administration of either CMT, however, had positive effects on the amount of bone and the biomechanical properties of rat femoral neck, but not the mineralization of the bone in the rat femoral neck. CONCLUSION: These data suggest that tetracycline derived antimetalloproteinase compounds can significantly and positively influence bone mechanical integrity associated with inhibition of collagen breakdown.

A cross sectional analysis of 5 different markers of collagen degradation in rheumatoid arthritis.

OBJECTIVE: To compare 5 different assays measuring collagen degradation in rheumatoid arthritis (RA). METHODS: Daily serum samples and 3 consecutive 24 h urine samples were obtained from 25 patients with RA and 20 control subjects. Levels of pyridinoline (PYD), deoxypyridinoline (DPYD), n-telopeptide (NTx), CrossLaps (XL), and carboxy-terminal peptide of type I collagen (ICTP) were determined by ELISA or radioimmunoassay. PYD, DPYD, NTx, and XL were measured in urine and expressed as a ratio of the amount of crosslink to mmoles of creatinine (Cr). ICTP was determined in serum. The day-to-day variability of urinary collagen crosslink levels and serum ICTP was assessed over 3 day hospitalization. RESULTS: Four of the 5 markers were significantly elevated in the RA cohort compared to controls: PYD (nmol)/Cr (median 33.8 vs 19.3; p = 0.0001), NTx (nmol)/Cr (median 22.5 vs 13.8; p = 0.01), XL (microg)/Cr (median 191.4 vs 126.1; p = 0.01), and ICTP (microg/l) (median 5.8 vs 3.7; p = 0.001). In the RA group, higher levels of the markers were associated with concomitant prednisone therapy. The levels of the 4 urine markers and of ICTP in serum exhibited little day-to-day variability. CONCLUSION: Biochemical evidence of increased collagen degradation can be readily observed in RA using simple quantitative assays. These measures have minimal short term, day-to-day variability and hence may be useful to assess the effect of potentially disease modifying therapies.