Estudio del metabolismo del complejo alfa2-macroglobulina y monofluorfosfato de sodio(MFP): relación entre dichos fenomenos y la mayor biodisponibilidad del fluor del MFP respecto del Fluoruro de Sodio / Study of the metabolism of the complex alpha2-macroglobulin and monophluorphosphate of sodium (MFP): relation between this phenomeno and the greater biodisponibility of the fluorine of the MFP respect of the sodium fluoride

El monofluorfosfato de sodio (MFP) es una droga que se prescribe como fuente de fluoruro para el tratamiento de la osteoporosis. El fluoruro es la especie mitogenica que estimula la diferenciacion y proliferacion de los osteoblastos. Posee mejor tolerancia gastrica que el NaF y se puede administrar con suplementos de calcio. Empiricamente se lo ha utilizado como equivalente del NaF aunque exhibe sustanciales diferencias farmacocineticas. Luego de una dosis oral del MFP, una pequeña fraccion de la misma se absorbe intacta y se liga a algunas globulinas del plasma formando un compartimiento de fluor no difusible (fluor ligado a proteinas), que no se observa cunado se administra NaF. El ion fluoruro no se liga a las proteinas del plasma. Cuando se comparan las curvas de fluoremia total en funcion del tiempo despues de la administracion de dosis equimolares de MFP y NaF se comprueba, en animales de experimentacion y en voluntarios humanos, que el area bajo la curva de MFP duplica la de NaF. Estos reaultados explican la reduccion a la mitad de la dosis terapeutica de fluor cuando se emplea MFP. El objetivo central de esta tesis fue develar el metabolismo del MFP para explicar la mayor tolerancia y eficiencia terapeutica, relacionadas con la mayor biodisponibilidad de fluor (respecto del NaF usado como standard). Accesoriamente surgio como un objetivo secundario identificar las caracteristicas de ligamiento de MFP a alfa2-M. La primera etapa de la investigacion se oriento a identificar las proteinas responsables de la aparicion del comportamiento de fluor ligado a proteinas. El MFP se liga principalmente a la alfa-globulinas y en menor grado a las beta-glñobulinas. El complejo MFP-globulina es estable y resiste cambios de pH, efecto de quelantes, dilucion o hidrolisis del MFP por fosfatasa alcalina. El ligamiento de MFP a las globulinas se aparta del modelo descripto por Scatchard de n sitios independientes y equivalentes. Los resultados experimentales sugieren un modelo de cooperativismo positivo. La alfa 2-macroglobulina (alfa2-M) es la proteina principalmente responsable de la fijacion de MFP. Al formarse el complejo MFP-alfa2-M, la proteina pierde su actividad como antiproteasa. El componente C3 del complemento, que comparte semejanzas estrurales con alfa2-M tambien liga MFP perdiendo su actividad biologica. La formacion del complejo MFP-proteinas con la concurrenteperdida de la actividad biologica de las proteinas participantes...(AU)

Therapeutic intervention with chicken egg white ovomacroglobulin and a new quinolone on experimental Pseudomonas keratitis.

BACKGROUND: Chicken egg white ovomacroglobulin (ovoM) is a potent protease inhibitor with broad-spectrum activity against various proteases. The combined effects of ovoM and the new quinolone, ofloxacin (OFLX) on experimental Pseudomonas aeruginosa keratitis were investigated. METHODS: The in vitro inhibitory effects of ovoM on protease activity in culture fluid of clinically isolated P. aeruginosa and on activity of human neutrophil elastase and cathepsin G were assayed using azo-casein as substrate. Albino rabbits received intrastromal injection of the isolated Pseudomonas strain (1 x 10(5) colony-forming units). At 16 h after inoculation, three treatment groups--0.1% ovoM alone, 0.3% OFLX alone, and a combination of both--and a non-treatment control group were tested. RESULTS: Protease activity in the culture solution and human neutrophil elastase was inhibited by ovoM, whereas cathepsin G was not inhibited effectively. In vivo additive therapeutic effects of ovoM and OFLX were observed at 96 h (P < 0.05 compared with OFLX alone). CONCLUSION: The results indicate that inhibition of proteolytic activity with ovoM is useful in preventing stromal degradation in P. aeruginosa keratitis.

Inhibitory effects of ovomacroglobulin on bacterial keratitis in rabbits.

We studied the inhibitory effects of chicken egg-white ovomacroglobulin (ovoM) on keratitis induced by 56,000-Da protease (56 KP) of Serratia marcescens and by elastase (PE) and alkaline protease (PAP) of Pseudomonas aeruginosa. The effects of ovoM on the serratial and pseudomonal keratitis in rabbits were also elucidated. In one model, four drops of 56 KP, PE, or PAP (1 mg/ml) were applied to wounded corneas of eight eyes. Thereafter, 80 microliters ovoM (10 mg/ml) was dropped into four eyes and 0.01 M phosphate-buffed 0.15 M saline (pH 7.4) into the other eyes as a control. The other in vivo test system involved intrastromal injection of S. marcescens or P. aeruginosa, by which each sample (10(5)-10(7) colony-forming units) mixed with ovoM was injected into one cornea and the other cornea received organisms without ovoM. OvoM completely inhibited the activity of these bacterial proteases in vitro and reduced corneal destruction in experimental keratitis in rabbits. In addition, greatly accelerated wound healing was observed.

Spreading of Serratia marcescens in experimental keratitis and growth suppression by chicken egg white ovomacroglobulin.

We studied the inhibitory effects of chicken egg white ovomacroglobulin, a broad-spectrum protease inhibitor, and a synthetic protease inhibitory peptide, Bz-GFR-O-mercaptoanilide, on the growth of two strains of Serratia marcescens in a synthetic medium. The growth of the virulent protease-producing strain, S. marcescens kums 3958 (kums 3958), was more rapid than that of the strain producing minimal protease, S. marcescens kums NA (kums NA), and kums 3958 was inhibited in a dose-dependent manner by ovomacroglobulin. The synthetic inhibitor also inhibited the growth of kums 3958 weakly. Dose-dependent enhancement of growth of kums NA was observed in the medium treated with the serratial 56 kilo Dalton protease. By immunohistochemical methods using an antibody to the bacteria, the spreading of kums 3958 was also studied in corneal tissue in experimental keratitis of the guinea pig. In vivo, kums 3958 grew locally, and then spread widely by destroying stromal tissue 7-8 hours after intrastromal injection of the organisms (3 x 10(4) colony-forming units). On the other hand, when kums 3958 mixed with ovomacroglobulin was injected into the cornea, the organisms remained locally in the corneal stroma which showed a lower grade of damage. These results indicate that proteases secreted from S. marcescens destroyed corneal stroma, yielding the organisms enough space for further spreading.

Serum alpha globulin fraction: survival-and-recovery effect in irradiated mice.

An alpha macroglobulin fraction (19S) was isolated from the serum of rats and BC(3)F(1) mice by zonal ultracentrifugation. Both the isologous and heterologous macroglobulin fractions increased survival among BC(3)F(1) mice x-irradiated with 750 roentgens. The mouse macroglobulin fraction also enhanced radiation recovery of hematopoietic tissue as measured by colony-forming assay and iron-59 incorporation into erythropoietic cells. The overall difference in hematopoietic activity in the irradiated (400 roentgens) mice treated with the macroglobulin fraction, in comparison with this activity in the controls, was three- to fivefold in the bone marrow and nine- to tenfold in the spleen between days 4 and 7 after irradiation. This effect was not obtained with the isologous serum protein fraction containing proteins of smaller molecular weight.