Britta Mansson-Rahemtulla

Specific assays for peroxidases in human saliva

The peroxidase activity in human whole saliva is due to salivary peroxidase and, in some cases, myeloperoxidase; it is usually determined by spectrophotometric methods based on the rate of oxidation of chromogen substrates. Thiocyanate ion, a normal component of saliva, interferes with these kinetic assays by competing with the chromogen for the available oxidizing equivalents; this results in underestimation of peroxidase activity. Both salivary peroxidase and myeloperoxidase will catalyse the peroxidation of the thiocyanate ion; the product, hypothiocyanite ion, is a reactive oxidizing agent. We have developed an assay for total peroxidase activity in saliva, based on the rate of formation of hypothiocyanite, which is not affected by the concentrations of thiocyanate found in saliva. Myeloperoxidase will catalyse the peroxidation of the chloride ion but salivary peroxidase will not; the product of this in neutral solution is the hypochlorite ion, which is also a reactive oxidizing agent. The specific contribution was determined of myeloperoxidase to total peroxidase activity in saliva by measuring the rate of both hypochlorite and hypothiocyanite formation. Because the thiocyanate ion will compete with the chloride ion, the concentration of thiocyanate in saliva samples must be reduced below 0.05 mM prior to measurements of the rate of hypochlorite formation.

Detection of the hypothiocyanite (OSCN−) ion in human parotid saliva and the effect of pH on OSCN− generation in the salivary peroxidase antimicrobial system

Human whole saliva contains the hypothiocyanite ion (OSCN-) which is the principal antimicrobial product of the salivary peroxidase system. The peroxidase system requires a source of peroxide in order to produce OSCN- and in the human mouth this source has been assumed to be primarily the peroxidogenic oral bacteria. However, we report here studies which show that samples of stimulated human parotid saliva collected directly from Stensons duct have concentrations of OSCN- which are similar to those found in human whole saliva. Thus, the peroxidogenic bacteria are not an absolute requirement for the generation of significant levels of OSCN- in the human mouth. Supplementation of human whole saliva with components [thiocyanite (SCN-), hydrogen peroxide (H2O2)] of the peroxidase system produces a 10-fold or greater increase in OSCN- concentration. However, the magnitude of this increase is critically dependent upon pH and upon the relative and absolute concentrations of SCN- and H2O2. The pH dependence of OSCN- generation is similar for human whole saliva and for the lactoperoxidase/SCN-/H2O2 system. The optimum is in the range 6.5-7.0. Samples of parotid saliva adjusted to pH 6.5 and supplemented with appropriate amounts of SCN- and H2O2 show increases in OSCN- concentrations which are similar to those observed with whole saliva. The results show that there is a significant source of H2O2 within the parotid gland, that the OSCN- generating potential of parotid saliva is similar to that of whole saliva and that the enhancement of OSCN- levels in saliva by addition of SCN- and H2O2 is critically dependent upon pH and upon the relative and absolute concentrations of H2O2 and SCN-.

Is thiocyanate peroxidation at equilibrium in vivo

The peroxidase-catalyzed oxidation of SCN- by H2O2 is an important in vivo reaction because it limits the accumulation of toxic H2O2 and provides significant concentrations of the antimicrobial agents, HOSCN and OSCN-. Data presented in this report suggest that the reaction: (Formula: see text) is in a state of dynamic equilibrium in vivo. Since OSCN- can form the weak acid HOSCN (pKa = 5.3), the equilibrium constant expression (Kox) for thiocyanate peroxidation is dependent on the concentration of hydrogen ions as well as the concentrations of H2O2, SCN-, HOSCN, OSCN- and water, and on the HOSCN ionization constant, Ka: (Formula: see text). The concentration of water is assumed to be constant and unaffected by the other components and is omitted from the Kox equation. The value of Kox was estimated from in vitro data to be 3.7 X 10(3) M-1 (S.D. = 0.8 X 10(3) M-1, n = 8). Using this value for Kox and observations of salivary concentrations of SCN- and HOSCN + OSCN- from several previous reports, the equilibrium concentrations of H2O2 in whole saliva were calculated to range from 8 to 13 microM. This range is consistent with reported estimates of 10 microM as the hydrogen peroxide tolerance limit for human cells.

Effects of Variations in pH and Hypothiocyanite Concentrations on S. mutans Glucose Metabolism

Hypothiocyanous acid (HOSCN) and hypothiocyanite (OSCN-) were generated by the antibody-independent salivary peroxidase (SP) system. The metabolism of Streptococcus mutans NCTC 10449 was examined by uniformly labeled glucose incorporation studies. We found that the SP-system causes a pH-dependent inhibition of 14C-labeled glucose uptake, and that the effects of HOSCN/OSCN- are bacteriostatic. The results also showed that, at low pH, bacteria required more time to recover fully from HOSCN/OSCN- inhibition. When control experiments were performed in the absence of HOSCN/OSCN-, but the pH was varied, we found a positive correlation between pH and the rate of 14C-glucose incorporation. The results also showed that pH did not affect the maximum incorporation of 14C-glucose, demonstrating that S. mutans can adapt to pH changes in the environment. Based on the data obtained, we postulate that the antibody-independent SP system plays an important role in the regulation of the metabolism of oral streptococci.

Human Salivary Peroxidase and Bovine Lactoperoxidase are Cross-reactive

Peroxidases are abundant in nature, and the primary function of mammalian peroxidases is to catalyze the peroxidation of halides and pseudohalides. Previous studies have shown that antibodies raised against bovine lactoperoxidase moderately cross-react with human salivary peroxidase, a feature that has been used in the present study to examine epitopes common to the antigen and human salivary peroxidase. Polyclonal antibodies against a highly purified preparation of bovine lactoperoxidase were raised in rabbits, and their properties were examined. In double-immunodiffusion experiments, the two enzymes showed partial identity, and in competitive radioimmunoassay and enzyme-linked immunosorbent assay, lactoperoxidase replaced the labeled and coated antigen, while salivary peroxidase did not. However, salivary peroxidase from human and rat saliva samples and the purified enzyme in its non-reduced, reduced, and de-glycosylated forms were recognized by these antibodies, as analyzed by Western blot analysis and immunodetection. The major activity of these antibodies was directed against the protein core of the antigen. Immunodetection of the peptide fragments of bovine lactoperoxidase and human salivary peroxidase revealed structural differences in the two enzymes. These antibodies also precipitated an in vitro translation product from rat-parotid-gland cell lysate that, on SDS-PAGE, compared favorably with the expected molecular weight of a de-glycosylated peroxidase. The antibodies partly inhibited the enzyme activity of salivary peroxidase and the peroxidase in rat parotid gland lysate, but the enzyme activity of lactoperoxidase was not affected by addition of anti-lactoperoxidase IgG between 25 and 400 wg/mL. The enzyme activity remained unchanged in all samples when pre-immune IgG was used.

Analyses of salivary components in leukemia patients receiving chemotherapy

We analyzed several salivary components in stimulated whole saliva from patients with acute leukemia who were undergoing chemotherapy. Saliva samples were collected at the time of diagnosis and longitudinally during the treatment period. Data analyses showed that patients with leukemia had significantly higher peroxidase and amylase activity and elevated concentrations of total protein at the time of diagnosis. After induction chemotherapy these parameters returned to normal values and remained constant during the observation period. At the time of diagnosis no significant differences in thiocyanate (SCN-) concentrations were found in saliva samples from control subjects and patients with leukemia. Treatment with cytotoxic agents resulted in granulocytopenia and a concomitant decrease in the SCN- concentration in saliva. The function of the salivary peroxidase system is impaired by the decrease in SCN- concentration, which may be a contributing factor to some of the oral complications that occur in patients undergoing chemotherapy.

Products of thiocyanate peroxidation: properties and reaction mechanisms

The lactoperoxidase-catalyzed oxidation of thiocyanate (SCN-) was studied in the pH range 3-8. The ultraviolet spectra of the oxidation products, the hypothiocyanite ion, OSCN- (at pH 8) and hypothiocyanous acid, HOSCN (at pH 3), were recorded. The absorbance maxima for OSCN- and HOSCN were observed at 220 and 240 nm, respectively. The extinction coefficients for OSCN- and HOSCN were determined to be 3870 (at 220 nM) and 95 M-1 X cm-1 (at 240 nM), respectively. Pure solutions of OSCN- (at pH 8) and HOSCN (at pH 3) were stable, but the mixtures of these two species at intermediate pH values were unstable. The decomposition could be divided into two periods, an initial period of rapid increase in oxidizing equivalents and a second period of decomposition. Decomposition during the second period followed first-order kinetics, and the pH-dependence of the apparent first-order rate constant was consistent with a decomposition mechanism which involved HOSCN. The first-order rate constant for this step was estimated to be 6 X 10(-3) s-1 at 37 degrees C.

A Mouthrinse which Optimizes in vivo Generation of Hypothiocyanite

We report here the properties of a mouthrinse which enhances one of the natural defense factors in human saliva, the salivary peroxidase system. Concentrations of the antimicrobial agent, the hypothiocyanite (OSCN-) ion, can be increased in vivo to bacteriostatic levels by use of a mouthrinse which is 4 mM (0.014%) in hydrogen peroxide and I mM (0.0097%) in potassium thiocyanate at pH 5.5. The volume of the rinse, the H2O2 concentrations, and the pH were shown to be determinants of the concentration of OSCN- generated by the rinse.

Isolation and characterization of bovine gingival proteoglycans versican and decorin

1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunological studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosylated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule.

Monolayer culture of rat parotid acinar cells without basement membrane substrates

SummaryAcinar cells have been difficult to maintain in primary or secondary cultures over extended periods of time. The most successful monolayer culture system reported to date requires basement membrane substrates. We report here a technique for culture of rat parotid acinar cells which does not rely upon basement membrane supports for maintenance and growth. The procedure involves gland excision, treatment to chelate metal ions, enzymatic digestion with collagenases and hyaluronidase, removal of fat and red blood cells by gravimetric separation, and nylon mesh filtration to yield a homogeneous suspension of small aggregates and single cells. The cells were examined for: a) morphology, identity, and growth; b) macromolecular synthesis; and c) secretory output. They were healthy, peroxidase positive, and growing for up to 10 d. Protein synthesis increased from the point of cell layer formation at 3 to 4 d, through 10 d, while DNA synthesis decreased. As in other studies, amylase secretion fell sharply between 2 and 4 d in culture and remained low. Although previous studies indicated that the initial isolation protocol left these acinar cells unable to thrive in monolayer culture except in the presence of basement membrane substrates, the modified technique reported herein allows these cells to attach, spread, and grow on a wide variety of commerically available plasticware. this method lends itself readily to long-term analysis of rat parotid acinar cell metabolism without the complications of dedifferentiation, cell loss through culture manipulation common in suspension cultures, or complex interactions between bioactive supports and cell surfaces.