Use of radioimmunoassay as a screen for antibiotics in confined animal feeding operations and confirmation by liquid chromatography/mass spectrometry.

Approximately one-half of the 50,000,000 lb of antibiotics produced in the USA are used in agriculture. Because of the intensive use of antibiotics in the management of confined livestock operations, the potential exists for the transport of these compounds and their metabolites into our nation's water resources. A commercially available radioimmunoassay method, developed as a screen for tetracycline antibiotics in serum, urine, milk, and tissue, was adapted to analyze water samples at a detection level of approximately 1.0 ppb and a semiquantitative analytical range of 1-20 ppb. Liquid waste samples were obtained from 13 hog lagoons in three states and 52 surface- and ground-water samples were obtained primarily from areas associated with intensive swine and poultry production in seven states. These samples were screened for the tetracycline antibiotics by using the modified radioimmunoassay screening method. The radioimmunoassay tests yielded positive results for tetracycline antibiotics in samples from all 13 of the hog lagoons. Dilutions of 10-100-fold of the hog lagoon samples indicated that tetracycline antibiotic concentrations ranged from approximately 5 to several hundred parts per billion in liquid hog lagoon waste. Of the 52 surface- and ground-water samples collected all but two tested negative and these two samples contained tetracycline antibiotic concentrations less than 1 ppb. A new liquid chromatography/mass spectrometry method was used to confirm the radioimmunoassay results in 9 samples and also to identify the tetracycline antibiotics to which the radioimmunoassay test was responding. The new liquid chromatography/mass spectrometry method with online solid-phase extraction and a detection level of 0.5 microg/l confirmed the presence of chlorotetracycline in the hog lagoon samples and in one of the surface-water samples. The concentrations calculated from the radioimmunoassay were a factor of 1-5 times less than those calculated by the liquid chromatography/mass spectrometry concentrations for chlorotetracycline.

Characterization of mixed micellar pseudostationary phases in electrokinetic chromatography using linear solvation energy relationships.

The influence of mixed micellar systems on retention and selectivity in micellar electrokinetic chromatography is examined using linear solvation energy relationships (LSER). Systems that were investigated include mixed bile salts [sodium deoxycholate (SDC) and sodium cholate (SC)] and mixed sodium dodecyl sulfate (SDS)-bile salt systems (e.g., SDS-SC and SDS-SDC). The retention behavior in individual and mixed micellar systems is primarily determined by size and hydrogen bond acceptor strengths of solutes. Through a comparative study of the LSER coefficients in the individual and mixed micellar systems, it was concluded that hydrogen bonding interactions have a significant effect on selectivity of these pseudostationary phases in electrokinetic chromatography. The interactive properties of the mixed micelles are different from the constituent individual micelles, however, the overall characteristics are closer to one of the bile salt micelles in the mixture even at the equimolar compositions.

Chemical selectivity in micellar electrokinetic chromatography. II. Rationalization of elution patterns in different surfactant systems.

Retention behavior in micellar electrokinetic chromatography (MEKC) is investigated using linear solvation energy relationships (LSERs) for two pseudo-stationary phases, one consisting of cationic micelles of tetradecyltrimethylammonium bromide (C14TAB) and the other of an anionic triblock copolymer, poly(methyl methacrylate-ethyl acrylate-methacrylic acid) (Elvacite 2669). It was found that solutes' migration behaviors in these two MEKC systems are mainly influenced by their size (V/100) and hydrogen bonding acceptor (HBA) strength (beta). However, solutes' hydrogen bonding donor (HBD) strength (alpha) has minor effects on their migration in MEKC. The characteristics of these two systems were compared to three other previously reported anionic micellar systems of sodium dodecyl sulfate (SDS) (anionic hydrocarbon), sodium cholate (SC) (anionic bile salt) and lithium perfluorooctane sulfonate (LiPFOS) (anionic fluorocarbon). It was concluded that hydrogen bonding interactions play a major role in providing different chemical selectivity among these five MEKC systems. Both C14TAB micelles and the ionic polymer of Elvacite 2669 provide hydrogen bonding acceptor (HBA) sites for solutes, which is similar to SC micelles. In fact, C14TAB is the strongest HBA, while Elvacite 2669 has HBA strength similar to that of SC micelles. On the other hand, the fluorocarbon micelles of LiPFOS are the strongest hydrogen bond donor (HBD) micelles, followed by the weak HBD SDS micelles. In general, cavity formation has little or no effect on chemical selectivity among hydrocarbon surfactant MEKC systems (i.e., SDS, SC and C14TAB). Information obtained from the LSER analysis is used to rationalize the elution patterns in MEKC with different types of pseudo-stationary phases.

Mixed micelles of short chain alkyl surfactants and bile salts in electrokinetic chromatography: enhanced separation of corticosteroids.

The separation of a complex mixture of 17 corticosteroids was investigated by mixed micellar electrokinetic chromatography (MMEKC) employing various bile salts and/or alkylsulfonates. In this study, influence of individual surfactants and mixed micelles of hydrocarbon-bile salt surfactants on retention behavior, selectivity and the size of the elution window is investigated. Retention behavior of corticosteroids in SDS and bile salt micelles is examined using linear solvation energy relationships (LSER). In addition, the effects of type of bile salt surfactant on elution patterns were investigated. It was found that separation patterns are mostly influenced by the number of hydroxyl functional groups on the steroidal backbone of the bile salts, while the type of ionic head group has little, if any, effect on the steroids separation. Comparisons between mixed micellar techniques and the inclusion of conventional modifiers to various single and binary surfactant systems were made. The addition of modifiers such as acetonitrile, urea and beta-cyclodextrin to SDS surfactant systems, as well as mixed bile salt systems of sodium taurocholate and sodium glycodeoxycholate, did not improve the separation of the steroids. On the other hand, the addition of the short-chain alkylsulfonate sodium butanesulfonate to the mixture of taurocholate and glycodeoxycholate greatly improved the separation of the 17 corticosteroids and provided a baseline separation of all solutes. The effects of carbon chain length and concentration of alkylsulfonate on capacity factor, selectivity, efficiency and the size of the elution window were investigated.

Quantitative structure-activity relationships studies with micellar electrokinetic chromatography. Influence of surfactant type and mixed micelles on estimation of hydrophobicity and bioavailability.

Applications of micellar electrokinetic chromatography (MEKC) in quantitative structure-activity relationships (QSAR) were studied. First, quantitative structure-retention relationships (QSRR), which describe the correlation between logarithm of capacity factor (log k') in MEKC and logarithm of distribution coefficient between 1-octanol and water (log P(ow)), were investigated for 60 aromatic compounds and 9 corticosteroids using three different anionic surfactants [e.g., sodium dodecyl sulfate (SDS), sodium cholate (SC), and lithium perfluorooctane sulfonate (LiPFOS)], one cationic surfactant (C14TAB), and mixed anionic micellar systems. Linear solvation energy relationships (LSER) and solvatochromic parameters were used to shed light on the different log k' vs. log P(ow) relationships of the various surfactants. It was concluded that hydrogen bonding interactions have a great influence on retention behavior in MEKC and its relationships with hydrophobicity. Interestingly, bile salt surfactants (e.g., SC) and mixed bile salt micellar systems provide better correlations for log k' vs. log P(ow) than SDS and/or SDS with buffer additives (e.g., beta-cyclodextrin, urea, and acetonitrile). Using SC micelles, only one line was adequate to describe the relationship between retention in MEKC and hydrophobicity for a group of 60 aromatic compounds. The existence of higher correlation for the SC system was attributed to a similar hydrogen bonding pattern between SC micelles and 1-octanol. In the SDS system, however, three lines were recognized for the congeneric subgroups of compounds. This is due to the hydrogen bond door (HBD) characteristic of SDS micelles that selectively differentiate between the solutes with different hydrogen bond acceptor (HBA) strength, thus demonstrating that retention is not solely based on hydrophobicity. A similar result was observed for a C14TAB-MEKC system, however, the HBA characteristic of C14TAB selectively differentiates between the solutes with different HBD strength. In addition, quantitative retention-activity relationships in MEKC were also investigated for 9 corticosteroids. Two types of biological activities [small intestinal absorption in the rat (log A/NA) and protein binding to human serum albumin (log B/F) were examined in this work. High correlations were observed between bioactivity and log k' in MEKC using bile salt surfactants and mixed bile salt systems.

Mixed micellar electrokinetic chromatography of corticosteroids.

Mixed micellar electrokinetic chromatography (MMEKC) using mixtures of bile salt surfactants and/or sodium dodecyl sulfate (SDS) was employed to separate a group of corticosteroids. Resolution of electrokinetic chromatography (EKC) can be greatly enhanced by the use of mixed micellar systems due to the fact that the composition of the mixed micelles has a great influence on retention, selectivity and the size of the elution window. By combining surfactants with different structural properties, solute-micelle interactions were manipulated in order to elicit specific separations. Various combinations of bile salts and/or SDS at different mole fractions as well as total micelle concentrations were used in order to enhance the resolution of corticosteroid separations. Large changes in retention and selectivity were observed that often resulted in frequent variations in elution order. In addition, the composition of mixed micellar systems had a great influence on the elution window in EKC, as measured by the ratio of tmc/teo. Addition of SDS to the mixtures of bile salt micelles resulted in significant extension of the elution window and subsequently improvement in resolution. A separation of seventeen corticosteroids was achieved. Finally, MMEKC was applied in order to separate the steroidal components of a mixture of three anti-inflammatory creams.

Lack of clinical significance of "enzyme-only" red cell alloantibodies.

In a retrospective study on samples from 10,000 recently transfused patients, 35 samples were found to contain an antibody that reacted with ficin-treated red cells but was not demonstrable by low-ionic-strength saline solution and indirect antiglobulin test (LISS-IAT). In those 35 patients, the specificity of the antibody was such that each patient would have been transfused with antigen-negative blood had the antibody reacted in LISS-IAT. Tests on red cells from the units already transfused showed that 19 patients had among them received, by chance, 32 antigen-positive and 74 antigen-negative units. The remaining 16 patients had among them received 57 units that were, again by chance, all antigen negative. One patient given antigen-positive blood suffered a delayed transfusion reaction; in two others the antibodies became LISS-IAT active after transfusion. However, similar changes to the LISS-IAT-active state were seen with two antibodies of patients given only antigen-negative blood. Also found in the 10,000 patients were 28 clinically insignificant antibodies, 77 sera in which the antibody was too weak to identify, and 216 autoantibodies that reacted only with ficin-treated red cells. These data support a belief, generally held in the United States but not necessarily elsewhere, that the use of protease-treated red cells for routine pretransfusion tests creates far more work than the accrued benefits justify.

Altered thyroid status regulates the adipocyte A1 adenosine receptor-adenylate cyclase system.

To assess the effect of hyperthyroidism on the adenosine receptor-adenylate cyclase system in adipocytes, membranes from hyperthyroid and control rats were prepared. Rats were rendered hyperthyroid by five days of injection with triiodothyronine (T3). Basal as well as isoproterenol-, sodium fluoride-, forskolin- and manganese (Mn++)-stimulated adenylate cyclase activities are attenuated 20-30% in adipocyte membranes from hyperthyroid animals. There is a greater inhibition of total adenylate cyclase activity in response to R-PIA, A1 selective inhibitory agonist, in membranes from hyperthyroid animals. However, on a percentage basis, R-PIA is equally effective at inhibiting adenylate cyclase activity in control and treated membranes. Using antagonist radioligands, [3H]XAC (A1 receptor) and [125I]CYP (beta-adrenergic receptor), no significant alteration in receptor number is observed in hyperthyroidism. In addition, no alteration in Gi protein-A1 receptor coupling is noted as exhibited by R-PIA competition curves. These findings suggest hyperthyroidism most likely results in a decrease of the catalytic moiety of adenylate cyclase either quantitatively or functionally.

Multiple components of the A1 adenosine receptor-adenylate cyclase system are regulated in rat cerebral cortex by chronic caffeine ingestion.

The effects of chronic caffeine on the A1 adenosine receptor-adenylate cyclase system of rat cerebral cortical membranes were studied. Caffeine treatment significantly increased the number of A1 adenosine receptors as determined with the A1 adenosine receptor antagonist radioligand [3H]xanthine amine congener (XAC). R-PIA (agonist) competition curves constructed with [3H]XAC were most appropriately described by a two affinity state model in control membranes with a KH of 2.1 +/- 0.8 and a KL of 404 +/- 330 nM with 50 +/- 4% of receptors in the high affinity state (%RH). In contrast, in membranes from treated animals, there was a marked shift towards the high affinity state. In three of seven animals all of the receptors were shifted to a unique high affinity state which was indistinguishable from the KH observed in membranes from control animals. In four of seven animals the %RH increased from 50 to 69% with KH and KL indistinguishable from the control values. Thus, the agonist specific high affinity form of the receptor was enhanced following caffeine treatment. Maximal inhibition of adenylate cyclase activity in cerebral cortical membranes by R-PIA (1 microM) was significantly increased by 28% following caffeine treatment, consistent with an increased coupling of receptor-Gi protein with adenylate cyclase. Importantly, the quantity of Gi (alpha i) in rat cerebral cortex, determined by pertussis toxin-mediated labeling, was also increased to 133% of control values by this treatment. Thus, multiple components and interactions of the A1 adenosine receptor-adenylate cyclase complex are regulated by caffeine. These changes are likely compensatory measures to offset blockade of A1 receptors in vivo by caffeine and lead to a sensitization of this inhibitory receptor system.

Comparative characterization of organic emissions from diesel particles, coke oven mains, roofing tar vapors and cigarette smoke condensate.

This paper reports the characterization of the extractable organics from diesel particulate emissions compared to other complex organics which have been reported to increase the risk of human lung cancer. Class fractions of diesel, cigarette smoke condensate, roofing tar, and coke oven extracts were obtained using liquid/liquid partitioning and silica gel chromatography. Capillary GC/MS was used to identify compounds in each extract fraction. This manuscript reports the mass distribution after fractionation of each extract, all identified fraction components and quantification of selected mutagenic and carcinogenic compounds.

Determination of human body burden baseline date of platinum through autopsy tissue analysis.

Results of analysis for platinum in 97 autopsy sets are presented. Analysis was performed by a specially developed emission spectrochemical method. Almost half of the individuals studied were found to have detectable platinum in one or more tissue samples. Platinum was found to be deposited in 13 of 21 tissue types investigated. Surprisingly high values were observed in subcutaneous fat, previously not considered to be a target site for platinum deposition. These data will serve as a human tissue platinum burden baseline in EPA's Catalyst Research Program.

Trace elements in hair, as related to exposure in metropolitan New York.

Previous studies have revealed that trace element concentrations in hair can reflect exposure in cases of frank poisoning and deficiency. This study reports significant correlations within a single metropolitan area between trace-element content of hair and exposure (as measured by analyses for the corresponding elements in dustfall or housedust) for Ba, Cr, Pb, Hg, Ni, Sn, and V. Age, sex, hair color, and smoking habits were factors included in the statistical evaluation. Several metals increase and decrease together in the hair specimens, in agreement with trends reported for other human tissues.