Metabolism studies, HPLC method

In a study of the metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes, a high performance liquid chromatography (HPLC) method has been developed that separates and quantitates methyl parathion and six of its hepatic biotransformation products (Anderson et al. 1992). The six biotransformation products identified are methyl paraoxon, desmethyl parathion, desmethyl paraoxon, 4-nitrophenol, />nitrophenyl glucuronide, and /wiitrophenyl sulfate. This method is not an EPA or other standardized method, and thus it has not been included in Table 7-1. 

By utilizing the HPLC method, it is possible to determine the level of each individual toxin in sample solutions. This provides a "toxin profile" that can be very useful in PSP toxin research studies. The ability to examine relative changes in toxin concentration and profile has greatly facilitated studies relating to toxin production by dinoflagellates, metabolism of toxins in shellfish, and movement of toxins up the food chain. Since the HPLC method is easily automated and requires only very small sample sizes (< 1 g tissue), it has clear advantages over other analytical procedures for the toxins in many research situations. Two examples of the utilization of HPLC for the study of the PSP toxins follow. 

The 3,4-dihydrodiol is a major component of the free dihydrodiols formed in mouse skin maintained in short-term culture (28). The optical purities of these dihydrodiols were determined by a CSP-HPLC method (43). The metabolic fates of the enantiomeric DMBA 3,4-dihydrodiols are not yet known. Studies in our laboratory indicate that the products formed in liver microsomal metabolism of DMBA 3,4-dihydrodiol bind extensively to the components of liver microsomes and the expected 1,2,3,4-tetrols of DMBA were not detected in the acetone/ethyl acetate extract of the incubation mixture (unpublished results). It is known that these products bind extensively to DNA... 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

The pharmacokinetics of rifaximin after oral administration has been studied in healthy volunteers and patients with intestinal infections or IBD. The aim of these studies was to confirm the low, if any, systemic absorption of the drug metabolism and excretion data are scant. In all these investigations a sensitive high-pressure liquid chromatographic (HPLC) method was used to measure rifaximin in body fluids. 

While consumption of products rich in resveratrol appears to be beneficial for the reasons discussed above, little is known about resveratrol bioavailability in humans and animals. Emilia et al. (1999) developed an analytical method to measure stilbene present in blood. Resveratrol administered orally to rats was detected in plasma. Excellent HPLC-based separation of tram-resveratrol from other compounds in the blood was achieved, allowing a rapid analysis of the sample for absorption, distribution, and metabolism studies. 

Thus, the analytical HPLC method for glycolipids is proving useful for a variety of studies related to glycosphingolipid function and metabolism. 

The study of sulfide metabolism at hydrothermal vents dictated the development of methods that could process hundreds of samples which contain complex mixtures of sulfur compounds in a variety of blood, seawater and tissues samples. In addition, we needed the capability of using "S-radiolabeled compounds for the tracing of complex sulfur metabolic pathways in bacteria and animal compartments of the different hydrothermal vent symbioses. In some instances, in situ sampling by submersibles at depths of 2500 meters with associated recovery times of two hours necessitated the remote derivatization of samples at depth prior to recovery. None of the above methods completely met our needs. We have adapted the bimane-HPLC method (24.351 for shipboard use and have found it a particularly robust method for studying a number of questions concerning the role of reduced sulfur compounds in the marine environment. 

We will present the basic requirements for preparing a bimane derivatized sample for HPLC analysis ana the preparation or appropriate blanks. We will then discuss the specific methods used for the three studies presented in the results section i) phytoplankton studies ii) sediment core studies iii) sulfide metabolism studies in invertebrate-bacteria symbiosis. 

A review of HPLC methods for antiepileptic drug analysis was published in 1987 by Juergens.18 Standard analytical separations were compared with narrow-bore separations, and a 70% reduction in the cost of solvents was possible, owing to a reduction in flow rate from 1 ml/min for the analytical column to 0.3 ml/min for the narrow-bore column. Gayden et al.19 developed an isocratic method, using narrow-bore columns, to quantify adenosine (Ado) release by dispersed rat renal outer medullary cells under conditions of normoxia and hypoxia. Standard HPLC with UV detection has been the predominant method for studying the metabolic pathways of adenosine and measuring the Ado breakdown products inosine (Ino) and hypoxanthine (Hyp). However, the conventional methods lack reliability... 

This chapter describes the use of the HPLC method to assay the activity of several enzymes simultaneously. The examples include several different enzymes that can use the same substrate and form the same product, a single enzyme that can use different substrates to form different products and two different activities using the same substrate to form different products. In another example the use of the HPLC method to study metabolic pathways is described through a series of reconstitution studies, and finally the HPLC method is applied to the anabolism of adenosine. 

Glutamine synthetase and glutamic acid decarboxylase catalyze the conversion of glutamic acid to glutamine and y-aminobutyric acid (GABA), respectively. Since both activities are involved in the metabolism of this amino acid, it would be useful to be able to study both simultaneously. And indeed, an HPLC method was developed to measure both activities in crude extracts. 

The study of cell culture supernatants and media was chosen to exemplify the utility of the optimized quaternary HPLC method. The rate at which amino acids are consumed by protein synthesis or other metabolic pathways can be quantified by performing amino acid analysis on supernatants of the protein producing cell cultures. Optimization of target protein expression can then be achieved by feeding the culture concentrated supplements rich in those amino acids that are rapidly consumed. 

Measurement of underivatized propranolol enantiomers in serum using a celluIose-tris(3,5-dimethylphenylcarbamate) high performance liquid chromatographic (HPLC) chiral stationary phase" (54). A method for the direct measurement of the enantiomers of propranolol in human serum was developed using the OD CSP and a mobile phase composed of hexane 2-propranol W,W-dimethyloctylamine (92 8 0.01, v/v/ v). The assay was validated for use in pharmacokinetic and metabolic studies and was subsequently used in the investigation of the effect of cimetidine on the metallism and clearance of propranolol enantiomers (60). 

These preclinical drug metabolism studies may also include metabolite profiling in plasma, selected tissues, urine, and bile to assess the distribution and disposition of potentially important metabolites, such as those having a level 5% or greater relative to the parent compound. Metabolite profiling requires a technique to separate the parent compound from metabolites and other endogenous compounds. For small organic molecules, HPLC is usually the method of choice. For macromolecules, gel or capillary electrophoresis techniques can be defined with sufficient resolution capability to separate the... 

Due to its availability, synthetic racemic ( )-ABA has been used in most metabolic studies. It is only in recent years that chiral HPLC columns have become available. With these, one can rapidly resolve the two enantiomers of ABA (or their methyl esters) [128-131], of PA and the l 4 -diols of ABA [132], and of 7 -OH-ABA [133]. These methods can also be used to determine the optical purity of ABA and its metabolites [134,135]. 

The NTP has a study in progress on "Aryl Amine Adducts in Blood as Indicators of Exposure" (NTP 1991a). In this study, blood samples from 100 workers will be analyzed for hemoglobin o-toluidine adducts. MBOCA will be used to develop an HPLC method for separation and isolation of mitochondrial or total aryl amine-DNA adducts. In addition, the in vitro activation of potential carcinogens will be studied, and a mathematical model for MBOCA distribution, metabolism, and adduct formation will be prepared. The overall objective of the project is to develop a more sensitive adduct isolation procedure to be used for biological monitoring. The contact person for this is K. Cheever(NTP 1991a). 

Accelerator mass spectrometry (AMS) is an ultrasensitive analytical method for radioactivity analysis. AMS offers 10 -10 -fold increases in sensitivity over LSC or other decay counting methods so that levels as low as 0.0001 DPM can be detected (Brown et al., 2005, 2006). AMS has been applied to mass balance determination, pharmacokinetic studies of total radioactivity, and measurement of chemically modified DNA and proteins in humans after the administration of a low radioisotope dose (approximately lOnCi/person for mass balance and drug metabolism studies) (Buchholz et al., 1999 Garner, 2000 Garner et al., 2002 Liberman et al., 2004 White and Brown, 2004). In addition, off-line HPLC-AMS has been explored for metabolite profiling after... 

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