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S R Hagen

Publications and source records attributed to S R Hagen.

8 recordsLinked to original sources
PubMed1997

Analysis of a monophosphoryl lipid A immunostimulant preparation from Salmonella minnesota R595 by high-performance liquid chromatography.

MPL immunostimulant, a 4'-monophosphoryl lipid A (MLA) preparation obtained from the lipopolysaccharide of Salmonella minnesota R595, is being developed for several clinical indications. MPL comprises a mixture of MLA congeners that contain 4, 5, and 6 fatty acids. In this paper, we report a new high-performance liquid chromatography (HPLC) method for analyzing the congener composition and purity of MPL. MPL is first derivatized with dinitrobenzyloxyamine (DNBA), resulting in incorporation of the dinitrobenzyl chromophore at the reducing end of all MLA congeners. DNBA-MPL is then analyzed by reversed-phase HPLC on a Waters NovaPak C18, 4 microns particle size, 300 mm x 3.9 mm column. Optimal separation of DNBA-MLA species is obtained using a linear gradient of 10% to 80% isopropanol-water (95:5, v/v), 5 mM tetrabutylammonium dihydrogenphosphate (TBAP), in acetonitrile-water (95:5, v/v), 5 mM.TBAP, over 45 min. A synthetic compound, corresponding to a hexaacyl MLA congener, is used for determination of the detector response factor, allowing the MLA content of MPL (i.e., purity) to be determined. Overall, this method provides better separation, higher sensitivity, and is faster and saler than previous methods used for the analysis of MPL.

Adjuvants, Immunologic↗
PubMed1996

Preservation of global cardiac function in the rabbit following protracted ischemia/reperfusion using monophosphoryl lipid A (MLA).

Monophosphoryl lipid A (MLA), a derivative of the minimal substructure of lipopolysaccharide (lipid A) possesses immunomodulatory activity of the parent lipid A yet enjoys reduced toxicity. It has previously been reported that pretreatment with MLA reduces myocardial infarct size and stunning in dogs following ischemia and reperfusion. The aim of this study was to evaluate the ability of monophosphoryl lipid A (MLA) to preserve global cardiac function and peripheral hemodynamics in a rabbit model of prolonged regional ischemia (90 min), and reperfusion (6 h). An evaluation of potential mechanisms by which MLA may preserve cardiac function was also undertaken. Single dose pretreatment with MLA (35 micrograms/kg i.v.) 24 h prior to ischemia resulted in significant improvement in left ventricular developed pressure, dP/dt, rate-pressure product and mean arterial pressure during reperfusion (P < 0.05 v control). Although in this model of prolonged ischemia MLA pretreatment did not reduce infarct size (54.5 +/- 11.4% in control v 63.3 +/- 8.3% in MLA, P = N.S.), evaluation of myocardial adenylate and adenosine catabolite pools at the end of ischemia indicated a preservation of ATP and ADP and a decreased production of downstream adenosine catabolites including inosine, xanthine and uric acid. Adenosine kinase, but not 5'-nucleotidase (5'-NTase) or adenosine deaminase activity determined following reperfusion was 76% and 60% higher (P < 0.05) in non-risk and post-ischemic myocardium of MLA pretreated rabbits compared with controls. Although there was a trend toward lower tissue myeloperoxidase activity in post-ischemic myocardium from treated rabbits, the results were not significantly different from control animals. These results suggest that a 24-h pretreatment with MLA, without further treatment during ischemia or reperfusion was associated with: (1) preservation of global myocardial function during reperfusion; (2) preservation of myocardial high energy adenylates and reduced formation of adenosine catabolites during ischemia; (3) elevated myocardial adenosine kinase activity. Increased recycling of adenosine to phosphorylated nucleotides may result from MLA's affect on adenosine kinase, which could explain the drugs effect on adenylate and adenosine metabolite pools.

5'-Nucleotidase↗
PubMed1995

Analysis of mycolic acids by high-performance liquid chromatography and fluorimetric detection. Implications for the identification of mycobacteria in clinical samples.

Mycolic acids from Mycobacterium phlei and M. bovis cell wall skeletons (CWSs) were analyzed by HPLC. After saponifying lyophilized CWSs in methanolic KOH, the mycolic acids were quantitatively extracted into chloroform. Aliquots of the CWS mycolic acid extracts were then derivatized prior to HPLC analysis with a UV reagent, p-bromophenacylbromide (PBPB), and three fluorescent reagents, 4-bromomethyl-6,7-dimethoxycoumarin, 4-bromomethyl-7-acetoxycoumarin and 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one. A synthetic alpha-branched carboxylic acid was derivatized with the same reagents and used as an internal standard along with the mycolic acids. The derivatized samples were analyzed by reversed-phase HPLC on a Waters Novapak C18, 4 microns particle size, 150 mm x 3.9 mm stainless-steel column. Two solvent systems were used: (1) methanol and methylene chloride with the column at 30 degrees C, and (2) methanol and isopropanol with the column at 50 degrees C. Detection sensitivity with the fluorescent reagents was 16-50 times greater than the sensitivity observed with PBPB-derivatized samples. Unique mycolic acid elution profiles for the two mycobacterial species could be achieved with each of the solvent systems and derivatization reagents tested. Thus, the HPLC analysis of pre-column derivatized mycolic acids was useful as a means of rapidly identifying mycobacterial species. Replacement of methylene chloride with isopropanol and PBPB with a fluorescent derivatizing reagent could increase the safety and sensitivity of the assay, and make it more useful for the clinical identification of mycobacterial infections.

Cell Wall↗
PubMed1976

Measurement of circulating thyroid microsomal antibodies by the tanned red cell haemagglutination technique: its usefulness in the diagnosis of autoimmune thyroid diseases.

Thyroid-microsomal antibodies were quantitated by a new technique utilizing tanned sheep red blood cells coated with human thyroid microsomal antigens. This haemagglutination assay (MCHA) correlated with the immunofluorescent antibody (FAB) but not with the thyroglobulin haemagglutination antibodies (TGHA) assay. Of forty-one patients with Hasmimoto's thyroiditis, thirty-nine (95%) were MCHA but only twenty-four (59%) TGHA positive. Titres were similar for the hypothyroid and euthyroid patients. Patients less than 20 years of age had either negative (50%) or low titre (less than 1:160) TGHA but 100% positive MCHA at titres greater than 1:1280. Of twenty-one patients with Graves' disease eighteen (86%) were MCHA and six (29%) TGHA positive. Of thirty-two patients without thyroid disease eleven (34%) were MCHA and/or TGHA positive. On the basis of family history and associated abnormalities, in eight of eleven, positive antibodies may have been due to subclinical Hashimoto's thyroiditis. Fourteen subjects of a control group (10%) were MCHA positive. Seven of ten examined had goitres. MCHA is a simple and quantitative test, useful in the diagnosis of autoimmune thyroid diseases.

Autoantibodies↗
PubMedDate not supplied

Precolumn phenylisothiocyanate derivatization and liquid chromatography of amino acids in food.

A precolumn phenylisothiocyanate derivatization method is described for the determination of amino acids in protein hydrolysates from a wide variety of complex food matrixes, with and without performic acid oxidation pretreatment. Analysis of samples that were not pretreated with performic acid was necessary since this pretreatment destroyed an average of 25% of the histidine and 87% of the tyrosine present in the food samples. This method is rapid and reproducible; coefficients of variation between duplicate analyses of the same food item were less than 5% for a majority of the amino acids. Occasionally, variation between duplicate analyses for histidine and tyrosine was greater than 10%. Recoveries of amino acids added to samples were in the 100% range.

Amino Acids↗