Iodometric Assay Research Articles

Lipoprotein oxidation and measurement of hydroperoxide formation in a single microtitre plate.

The oxidative modification of lipoproteins is of clinical importance because of potential contribution to atherogenesis [1, 2, 3]. An early step in the complex process of oxidation is the peroxidation of polyunsaturated fatty acids. We describe here a method for the Cu(II)-catalyzed oxidation of human low density lipoproteins with the subsequent analysis of hydroperoxides formation in a single microtitre plate. The procedure includes a modification of an iodometric peroxide assay for test tubes using a commercially available reagent. The microtitre plate method correlated well with the test tube procedure (r = 0.99) and showed comparable sensitivity and reproducibility. It was sensitive down to 0.5 nmol hydroperoxides/well and linear up to at least 20 nmol well-1. The method can handle several hundreds of samples a day with considerably less labour than the test tube procedure. It was well suited to monitor the kinetics of lipoprotein oxidation. The method was also used to test the potency of antioxidants, however, some antioxidants may interfere with the iodometric reaction and should be tested in the assay before use.

Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe 2+ to Fe 3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe 3+-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 × 10 4 m −1 cm −1 consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay.

Simple and specific test for measuring lipid peroxides in plasma.

The specificity of an iodometric assay for measuring lipid peroxides in lipoproteins was tested, compared with the fluorimetric thiobarbituric acid assay, and adopted for detecting lipid peroxide in plasma samples. Oxidation of low density lipoproteins in vitro by Cu2+, lipoxidase, and phagocytosing polymorphonuclear leucocytes was sensitively detected by the iodometric assay. Unlike the thiobarbituric acid assay, neither non-lipid substances commonly present in plasma, nor platelet or polymorphonuclear leucocyte by-products interfered with the iodometric assay. The iodometric assay measured a normal mean (SD) plasma lipid peroxide concentration of 10.8 (2.1) microM; n = 63. Two weeks after the start of a high cholesterol diet in rabbits (n = 5), a sixfold increase in plasma lipid peroxide concentrations was measured by iodometric assay. The specificity of a simple and sensitive iodometric test of lipid peroxidation was superior to that of the thiobarbituric acid assay. This iodometric assay should therefore provide a much more accurate assessment of lipid peroxide in plasma samples.

REACTIVITY OF PHOTOCHEMICALLY‐GENERATED LIPID HYDROPEROXIDES IN CELL MEMBRANES WITH GLUTATHIONE PEROXIDASE

The ability of glutathione peroxidase (Gpx) to catalyze the reductive inactivation of photochemically-generated lipid hydroperoxides (LOOHs) was investigated, using hematoporphyrin derivative (HPD) as a photosensitizing agent and erythrocyte ghosts as membrane targets. Glutathione peroxidase was reactive toward photoperoxidized membranes only after their exposure to phospholipase A2 (PLA2). Iodometrically-determined LOOH values were typically 30-40% greater than values measured by enzymatic assay using Gpx and glutathione reductase. A consistent result was obtained when photooxidized membranes were treated with PLA2 and GSH/Gpx followed by iodometric assay, viz. persistence of approximately 40% of the starting LOOH. Whereas photooxidized egg phosphatidylcholine liposomes underwent total LOOH loss when incubated with PLA2 and GSH/Gpx, no net loss was observed with photooxidized cholesterol/dimyristoyl-phosphatidylcholine liposomes. The results suggest that cholesterol hydroperoxides in ghost membranes account for the Gpx-resistant fraction of LOOHs.

Stability of Cephalexin Monohydrate Suspension in Polypropylene Oral Syringes

The stability of cephalexin monohydrate suspension in plastic oral syringes was studied. Commercially available cephalexin monohydrate powder for oral administration was reconstituted according to the manufacturer's instructions and stored in the original containers or drawn into 5-mL clear polypropylene oral syringes. The original containers and syringes were divided into groups and stored at -20, 4, 25, 40, 60, or 80 degrees C. Powder from two additional lots was similarly reconstituted and packaged; these original containers and syringes were stored at 80 degrees C only to assess interlot variability. Immediately after reconstitution and at specified times during storage, three syringes and the corresponding three original containers stored at each temperature were removed, and their contents were analyzed for cephalexin concentration using the standard USP iodometric assay for antibiotics. The stability-indicating nature of the assay was documented. Cephalexin monohydrate followed a first-order rate of degradation at temperatures of 40, 60, and 80 degrees C. At temperatures of -20, 4, and 25 degrees C, cephalexin monohydrate exhibited no appreciable degradation during the 90-day study period. Cephalexin monohydrate suspension reconstituted from powder as a suspension and repackaged in clear polypropylene oral syringes was stable for 90 days when stored under ambient, refrigerated, and frozen conditions.

Detection of beta-lactamase activity among clinical isolates of Branhamella catarrhalis with six different beta-lactamase assays.

A total of 74 different clinical isolates of Branhamella catarrhalis were examined for their ability to produce beta-lactamase by six different beta-lactamase assays. These included a conventional tube and disk test, in which the chromogenic cephalosporin nitrocefin was used as a substrate; a disk procedure, in which pyridinium-2-azo-p-dimethylanaline cephalosporin was used as a substrate; broth and disk acidometric methods; and a conventional tube iodometric assay. A total of 58 of the study isolates produced beta-lactamase. In all cases, positive results were obtained with the nitrocefin tube and disk assays after 1 min. With the pyridinium-2-azo-p-dimethylanaline cephalosporin disk test, 57 of the 58 beta-lactamase-producing strains yielded a positive reaction in 1 min; the remaining strain was positive after 10 min. None of the beta-lactamase-producing strains produced positive reactions by either the broth or disk acidometric methods after 1 min. With the broth test, 10 min was required for positive test results for 42 strains; 30 min was necessary for 16 strains. By the disk acidometric procedure, all 58 strains were positive after 10 min. Of 58 beta-lactamase-producing strains, 30 were positive by the iodometric assay after 1 min, 13 strains required 10 min, and 4 strains were detected as being beta-lactamase positive only after 30 min. One beta-lactamase-producing strain remained negative by the iodometric method. Among the 16 strains of B. catarrhalis that lacked beta-lactamase that were examined in this study, no false-positive results were obtained by any of the six assays.

Stability of dicloxacillin sodium oral suspension stored in polypropylene syringes

The stability of dicloxacillin sodium in oral suspension stored in clear polypropylene oral syringes was studied. Commercially available dicloxacillin sodium powder for oral suspension from a single lot was reconstituted according to the manufacturer's instructions and drawn into 5-mL clear polypropylene oral syringes. The syringes were divided into groups and stored at -20, 4, 25, 40, 60 or 80 degrees C. Two additional lots were similarly reconstituted, repackaged, and stored at 80 degrees C only to assess interlot variability. Powder in the original containers was similarly reconstituted according to the manufacturer's instructions, and the containers were divided into groups and stored with the syringes. Immediately after reconstitution and at specified times during storage, three syringes and the original containers at each storage temperature were removed, and their contents were analyzed for dicloxacillin sodium concentration using the Standard USP Iodometric Assay. Dicloxacillin sodium follows a first-order rate of degradation at temperatures of 40, 60, and 80 degrees C. The rate of degradation changes to a zero-order process at temperatures of 25, 4, and -20 degrees C. At all temperatures, degradation occurred more rapidly when the drug was repackaged into unit dose polypropylene oral syringes than in the manufacturer's original container. Dicloxacillin sodium reconstituted from powder as oral suspension and repackaged in clear polypropylene syringes was stable for no longer than 7, 10, and 21 days when stored under ambient, refrigerated, and frozen conditions, respectively.

Inhibition of beta-lactamases from Yersinia enterocolitica by plant extracts.

Strain IP134 of Yersinia enterocolitica, which produces two chromosomal-mediated beta-lactamases, was used to detect the presence of beta-lactamase inhibiting agents in plants. Aqueous and alcoholic extracts were obtained from the aerial parts of 179 phanerogamous species, belonging to 39 botanical families. In an assay to detect synergy, eight plants representing a wide taxonomic distribution showed beta-lactamase inhibitory activity. An iodometric assay confirmed the inhibition of beta-lactamases in six of these plants, and revealed beta-lactamase inhibition to be masked by antibacterial activity in two additional plant extracts. Thus, beta-lactamase inhibitory activity was present in 4.5% of the tested species, 1.1% of which had simultaneous antibacterial activity detectable.

Rapid identification of Escherichia coli transformed by pBR322 carrying inserts at the PstI site

An iodometric assay for β-lactamase has been employed for identifying colonies of Escherichia coli transformed to tetracycline resistance ( Tc r ) by pBR322 carrying inserts at the PstI site. This assay is based upon the ability of β-lactamase produced by ampicillin-resistant ( Ap r ) cells to convert penicillin to penicilloic acid which in turn binds iodine. Growth and selection of E. coli transformed to Ap r Tc r or Ap s T r are obtained on Luria agar plates containing soluble starch and tetracycline. When indicator solution containing penicillin and iodine is added to the colonized plates, β-lactamase-producing ( Ap r ) colonies rapidly clear the overlying indicator solution whereas non-β-lactamase-producing ( Ap s ) colonies exhibit no clearing effect. This reaction persists and substantial numbers of viable cells remain well beyond the end of the 15-min observation period. In post-test assessment of phenotype, all nonclearing colonies exhibited the Ap s Tc r phenotype while those that cleared the indicating solution exhibited the Ap r Tc r phenotype. Application of this assay to an actual transformation experiment permitted rapid and unambiguous identification of the Ap s Tc r phenotype.

Nuclear magnetic resonance spectrometric assay of beta-lactamase.

Beta-Lactam antibiotics and the crude enzyme were mixed in deuterium oxide and placed in a nuclear magnetic resonance tube. The change of the nuclear magnetic resonance spectrum during the enzymatic reaction was then analyzed to determine beta-lactamase activity. By using beta-lactam antibiotics such as penicillins, cephalosporins, and cephamycins as substrates, a comparison of the beta-lactamase activities was made between the nuclear magnetic resonance spectrometric assay and the iodometric assay. There was a close correlation between these two methods.

Stability of frozen solutions of cefamandole nafate

The chemical, microbiological and visual stability of frozen solutions of cefamandole nafate was studied. Solutions of cefamandole nafate were prepared by diluting 1 g of drug with 3 ml of Water for Injection, USP, or 0.9% Sodium Chloride Injection, USP, or 5% Dextrose Injection, USP (i.m. dilutions); or with 50 or 100 ml of the latter two diluents (i.v. dilutions). Stability of samples stored in glass and polyvinyl chloride plastic containers for up to 52 weeks at -10 and -20 C was measured by microbiologic, polarographic, iodometric, nephelometric and chromatographic assay and pH was measured. In mice, LD50 tests were performed using the i.m. dilutions. I.M. dilutions of cefamandole nafate were stable for 52 weeks when stored at -20 C; at -10 C, however, some samples did not freeze completely and were turbid when thawed. I.V. dilutions were stable for 26 weeks when stored at -20 C. I.V. dilutions with D5W stored at -10 C developed a transient haze. A gradual decrease in pH, which was a function of storage time, was noted for the frozen solutions. Six months of freezing did not alter the LD50 in mice. Solutions of cefamandole nafate are stable for at least 26 weeks when stored at -20 C in glass or PVC containers.

Identification of an Imidazolinium salt, the major Product from Reaction of Benzathine with Iodine

The isolation and identification of an imidazolinium salt are described. Unambiguous determination of structure was accomplished by independent synthesis. The isolated product interferes in the iodometric assay of the benzathine salts of penicillin.

Problems with Iodometric Assay of Penicillin V Benzathine

Problems with Iodometric Assay of Penicillin V Benzathine

Iodometric assay method for beta-lactamase with various beta-lactam antibiotics as substrates.

The rapid fixed-time assay for penicillinase was modified for measuring beta-lactamase activity with twelve substrates, i.e., benzylpenicillin, ampicillin, cloxacillin, methicillin, carbenicillin, cefazolin, cephalothin, cephaloglycin, cephalexin, cephalosporin C, 7-aminocephalosporanic acid, and cefoxitin. The method depends upon the reduction of iodine by the hydrolyzed substrate. Determined experimentally, 1 mol of hydrolyzed penicillins consumed 3.4 to 4.0 mol of iodine (I(2)). Iodine consumption of hydrolyzed cephalosporins varied widely from 1.7 for cephalothin to 3.7 for cefazolin. The method is useful for routine assay of beta-lactamase activity with various substrates.

347 β-LACTAMASE ACTIVITY & ANTIMICROBIAL SUSCEPTIBILITY OF HEMOPHILUS INFLUENZAE AND PARAINFLUENZAE IN MICH

The incidence of β-lactamase activity of 1000 Hemophilus strains collected Sept 76-Jan 78 from pediatric and adult patients throughout Michigan (utilizing an iodometric assay, AAC 7:265:1975) was 8/52 (15%) for type b, 29/318 (9%) for non-type b H. influenzae and 31/630 (5%) for H. parainfluenzae. From Sept 76-July 77 H. influenzae type b isolates were recovered from 16 children with meningitis; none were positive for β-lactamase activity. Subsequently 4/7 CSF isolates were positive for β-lactamase activity. The in vitro susceptibilities of 30 β-lactamase (+) Hemophilus strains to ampicillin, chloramphenicol, cefamandole, cefachlor, tetracycline and trimethoprim-sulfamethoxazole were evaluated using a microtiter broth dilution method. 70% of strains were resistant to [amp] of ≥10 μg/ml (MIC range = 4-128 μg/ml), while all were inhibited by ≥1 μg/ml of chloramphenicol (MIC=0.25-1 μg/ml). Strains were highly susceptible to cefamandole (90% inhibited by ≥0.5 μg/ml), although only 25% of strains were inhibited by ≥1 μg/ml of cefachlor (MIC-1-16 μg/ml). All isolates were inhibited by [tetracycline] of ≥1 μg/ml. MICs of TMP-SMZ ranged from .0037/.148-.125/2.37 μg/ml with 75% of strains susceptible to ≥0.03/.59 μg/ml. The incidence of β-lactamase (+) Hemophilus strains in Michigan appears to be increasing. These strains are highly susceptible in vitro to chloramphenicol, cefamandole and TMP-SMZ.

Effect of Media Composition on the Penicillin Production

The conventional penicillin fermentation medium is composed of corn steep liquor, glucose, lactose, minerals, oil, and precursor. The penicillin activity was not affected, due to the addition of carbonate 0-1% or whale oil 0.5% instead of 1%. Also the omission of cupric, magnesium, manganese, zinc sulphates, and acetic acid did not affect the penicillin activity, while the omission of ammonium nitrate and potassium dihydrogen phosphate decreased the penicillin activity in the medium. The penicillin activity of a medium containing 2% calcium superphosphate was higher than that of the control medium, containing 0.4% potassium dihydrogen phosphate and 1% calcium carbonate. Instead of adding the precursor twice, after 0 and 48 hrs, the addition of phenylacetamide in the amount of 0.2%, at the start, did not affect the activity, while addition of phenylacetic acid (0.2%), at the start, decreased the penicillin activity. The omission of the precursors in the medium decreased the penicillin activity measured microbiologically, however, 6-aminopenicillanic acid content was relatively higher as compared with that of the control medium, containing precursor. These results were confirmed, using iodometric assay and paper chromatographic analysis.

Comparison of assay techniques for β-lactamase activity

Two chemical, one physical and two microbiological techniques for the assay of β-lactamase activity have been compared using three partially purified β-lactamases of different specificities. Seven cephalosporins and three penicillins with a wide range of susceptibilities have been used as substrate. The different methods gave results that were in general agreement with the exception of the hydroxylamine assay. The cephalosporins were most rapidly assayed by the uv method. Its suitability for use in the assessment of new cephalosporin analogues was confirmed, but it could not be used for penicillins. The iodometric assay was rapid and reliable but required large amounts of substrate. Where the amount of substrate available was limited, the microbiological techniques were preferred. The results obtained for enzyme activities against two of the cephalosporins and against the penicillins were higher with the hydroxylamine assay than with the other methods and it had no apparent advantage.

The use of -lactamases in the clinical laboratory.

Summary Eleven β-lactamases were tested for their ability to destroy the currently available β-lactam antibiotics. The biochemical activities of the enzymes against cephaloridine and benzylpenicillin were compared by iodometric assay. Their biological activity was tested by mixing various amounts of enzymes and of 13 different β-lactam antibiotic preparations and assaying the residual antibiotic by an agar-diffusion method, with Sarcina lutea as the test organism. All the enzymes destroyed 500 μg per ml of benzylpenicillin and the more potent ones also destroyed “penicillinase-resistant” penicillins and cephalosporins. Crude enzyme extracts from a strain of Enterobacter aerogenes and of Klebsiella aerogenes compared favourably with the bacillus enzymes in their action on cephalosporins. Suitable β-lactamases can therefore be used to destroy all the available β-lactam antibiotics in specimens from patients, either in blood cultures or to allow the assay of other antibiotics in patients being treated concurrently with both.

The β-Lactamases of Gram-Negative Bacteria and their Possible Physiological Role

Publisher Summary β -lactamases are the enzymes that are capable of hydrolyzing the β -lactam bond of penicillins and cephalosporins. The reaction catalyzed by β -lactamases with penicillins as substrates includes the rupture of the β -lactam bond to form the corresponding penicilloic acid. However, there is complexity in breakdown of the cephalosporins after β -lactamase action as there is no sporin and the formation of any single product—a fact that invalidates the iodometric assay of cephalosporinase for absolute measurement. Despite the wide and varying specificity, β -lactamases often require the 4-membered azetidinone ring to be condensed either with a thiazolidine (penicillins) or a dihydrothiazine nucleus (cephalosporins). The molecular basis of the resistance of Gram-negative bacteria to β -lactam antibiotics is very different from their Gram-positive counterparts. Some of the possible physiological role recognized for β -lactamases includes—namely, (1) hydrolysis of β -lactam antibiotics when required and (2) destruction of β -lactam antibiotics. The role of protecting the organisms that produce β -lactamases against the deleterious effects of penicillins and cephalosporins can often provide a major obstacle to the successful use of β -lactam antibiotics for therapy.

Activity of penicillinase in Staphylococcus aureus as studied by the iodometric method.

Among naturally occurring strains of Staphylococcus aureus, resistance to penicillin G is observed only in those that produce penicillinase [1]. Strains lacking this enzyme are invariably highly sensitive to the action of penicillin. Elaboration of penicillinase thus appears to be the only clinically important mechanism of resistance to penicillin in these organisms [2]. A test for the activity of penicillinase that is rapid and simple enough to be employed in the clinical bacteriology laboratory would be useful as a guide to antibiotic therapy, since it would detect the substance actually responsible for resistance to penicillin. Several tests for detection of penicillinase have been devised [3-14], but most of them are too cumbersome or too time-consuming for routine hospital use. A simple plate test based on the iodometric assay originally described by Ferret [10] circumvents most of the disadvantages inherent in other methods. This test has been used by Anderson and Lewis in the investigation of resistance to ampicillin in Salmonella typhimurium [15], and by Novick and Richmond in genetic studies on S. aureus [16], but to our knowledge has not been employed for routine work in hospital bacteriology. This study was undertaken to investigate the usefulness of this test in the evaluation of strains of