Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.
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Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen. (+info)
Isolation and characterization of plasmin-generated bioactive fragments of IGFBP-3.
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Insulin-like growth factor-binding protein-3 (IGFBP-3) was digested with plasmin, and the proteolytic fragments were isolated by HPLC and tested for bioactivity as measured by stimulation of glucose uptake in microvessel endothelial cells. Two of the pooled fractions of the digest stimulated glucose uptake. The major bioactive pool, at an estimated protein concentration <50 ng/ml, stimulated glucose uptake to 150% of control with greater stimulation and 220% of control at approximately 250 ng/ml. Two fragments were present in the bioactive fraction, the dominant one migrating at approximately 20,000 and the other at approximately 8,000. Both fragments bound 125I-labeled insulin-like growth factor and [3H]heparin. NH2-terminal amino acid analysis of the bioactive peak yielded two sequences. One, representing the majority of the material, had an NH2-terminal sequence identical to IGFBP-3; the second fragment began at amino acid 202 of IGFBP-3. In contrast to the bioactive fragments, intact IGFBP-3, at concentrations up to 130 microgram/ml, had no bioactivity. These findings demonstrate that IGFBP-3 can be degraded into fragments that have potent bioactivities that are not present in the intact IGFBP-3 molecule. (+info)
Nonsaturable entry of neuropeptide Y into brain.
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Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB. (+info)
Kinetics of dodecanedioic acid triglyceride in rats.
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The kinetics of the triglyceride of dodecanedioic acid (TGDA) has been investigated in 30 male Wistar rats after a rapid intravenous bolus injection. TGDA and its product of hydrolysis, nonesterified dodecanedioic acid (NEDA), were measured in plasma samples taken at different times using an improved high-performance liquid chromatographic method. The 24-h urinary excretion of TGDA was 1.54 +/- 0.37 micromol, corresponding to approximately 0.67% of the administered amount. Several kinetics models were considered, including central and peripheral compartments for the triglyceride and the free forms and expressing transports between compartments with combinations of linear, carrier-limited, or time-varying mechanisms. The parameter estimates of the kinetics of TGDA and of NEDA were finally obtained using a three-compartment model in which the transfer of TGDA to NEDA was assumed to be linear, through a peripheral compartment, and the tissue uptake of NEDA was assumed to be carrier limited. TGDA had a large volume of distribution ( approximately 0.5 l/kg body wt) with a fast disappearance rate from plasma (0.42 min-1), whereas NEDA had a very small volume of distribution ( approximately 0.04 l/kg body wt) and a tissue uptake with maximal transport rate of 0.636 mM/min. In conclusion, this first study on the triglyceride form of dodecanedioic acid indicates that it is rapidly hydrolyzed and that both triglyceride and nonesterified forms are excreted in the urine to a very low extent. The tissue uptake rate of NEDA is consistent with the possibility of achieving substantial energy delivery, should it be added to parenteral nutrition formulations. Furthermore, the amount of sodium administered with the triglyceride form is one-half of that necessary with the free diacid. (+info)
The pharmacokinetics of artemisinin after administration of two different suppositories to healthy Vietnamese subjects.
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Eight healthy Vietnamese male subjects received 400 mg artemisinin formulated into fatty suppositories (FS), and six different subjects received 500 mg of artemisinin formulated in polyethylene glycol suppositories (PEGS). Plasma concentrations were measured by high-performance liquid chromatography with electrochemical detection; concentration versus time curves were analyzed with nonparametric methods. No statistically significant differences were found between the two formulations. The maximum concentration (Cmax) was 100 +/- 102 microg/L (mean +/- SD, range = 24-330) microg/L (FS), the pharmacokinetic lag time (Tlag) was 1.3 +/- 1.0 hr (range = 0-3) (FS), and the time of the maximum concentration (Tmax) was 7.1 +/- 2.1 hr (range = 3-10) hr (FS). Because artemisinin is not available for intravenous dosage, absolute bioavailability cannot be assessed. However, compared with a previous study on oral artemisinin in healthy Vietnamese subjects, bioavailability relative to oral administration was estimated to be approximately 30%. We conclude that therapeutic blood concentrations of artemisinin can be reached after rectal dosage. The dose after rectal administration should probably be higher than after oral administration; doubling or tripling the oral dose might be necessary, which would imply a rectal dose of at least 20 mg/kg of body weight given twice a day. (+info)
Relationships between the lipophilicity of some 1,4-piperazine derivatives of aryloxyaminopropanols and their beta-andrenolytic activity.
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Nineteen 1,4-piperazine derivatives of aryloxyaminopropanol were evaluated with respect to beta-adrenolytic activity. The retention factors obtained from HPLC, RM values obtained from partition TLC and the lipophilic Hansch's (4) constants pi were determined and the compounds were studied with respect to their lipophilicity based on chromatographic properties. The study of the influence of different substituents introduced at the para position on the phenyl ring on the retention factor indicated the log k vs. the number of carbon atoms in R1 substituent to be a linear relationship. Attempts have been made to relate the beta-adrenolytic activity to the lipohydrophilic parameters by deriving a quantitative relationship between them. Significant parabolic correlation was observed between the beta-adrenolytic activity and the logarithm of the retention factor, log k. An analogous relationship was obtained between the beta-adrenolytic activity of the compounds and the RM values obtained from partition TLC as well as Hansch's lipophilic constants pi. (+info)
Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.
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Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100. (+info)
Rapidly available glucose in foods: an in vitro measurement that reflects the glycemic response.
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BACKGROUND: A chemically based classification of dietary carbohydrates that takes into account the likely site, rate, and extent of digestion is presented. The classification divides dietary carbohydrates into sugars, starch fractions, and nonstarch polysaccharides, and groups them into rapidly available glucose (RAG) and slowly available glucose (SAG) as to the amounts of glucose (from sugar and starch, including maltodextrins) likely to be available for rapid and slow absorption, respectively, in the human small intestine. OBJECTIVE: We hypothesize that RAG is an important food-related determinant of the glycemic response. DESIGN: The measurement of RAG, SAG, and starch fractions by an in vitro technique is described, based on the measurement by HPLC of the glucose released from a test food during timed incubation with digestive enzymes under standardized conditions. Eight healthy adult subjects consumed 8 separate test meals ranging in RAG content from 11 to 49 g. RESULTS: The correlation between glycemic response and RAG was highly significant (P < 0.0001) and a given percentage increase in RAG was associated with the same percentage increase in glycemic response. After subject variation was accounted for, RAG explained 70% of the remaining variance in glycemic response. CONCLUSIONS: We show the significance of in vitro measurements of RAG in relation to glycemic response in human studies. The simple in vitro measurement of RAG and SAG is of physiologic relevance and could serve as a tool for investigating the importance of the amount, type, and form of dietary carbohydrates for health. (+info)