High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes

A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.     

A gel filtration high-performance liquid chromatographic method for determination of hepatic and renal metallothionein of rat and in comparison with the cadmium-saturation method

Metallothionein (MT) is a low-mol-wt protein. The essential trace elements copper and zinc and the potentially toxic elements, such as cadmium, can induce the synthesis of and bind to MT. The major functions of MT are related to metal metabolism. This paper reported and evaluated a new method for determination of hepatic and renal MT of rat by using high performance liquid chromatography (HPLC) with a Superdex 75 gel filtration column. The tissue was homogenized and centrifuged, then the supernatant was pretreated simply by cadmium saturation and heating before HPLC determination. The MT was completely and clearly separated from other proteins in the rat tissues in a short time, and was quantitated directly as a function of UV absorbance at 250 nm. The recovery both for hepatic and renal MT of rat were exceed 90%. The coefficient of variation was 1.3% for hepatic MT of rat and 1.7% for renal MT of rat. The detection limit was 0.265 μg for hepatic MT and 0.095 μg for renal MT of rat. The present method was compared with the traditional cadmium-saturation method for determination of hepatic and renal MT of rat. A good correlation was found in these two methods.     

Growth and reproduction in higher plants I. Theoretical analysis by mathematical models

To analyse the whole life of higher plants, an attempt was made to describe their growth and reproduction by mathematical models based on the elements determining matter production and economy of the matter. A plant body was regarded as a compound system of two parts; “productive part” and “reproductive part”. A parameter (reproductive index) was introduced to connect these two parts, and a set of the mathematical models describing the quantitative growth of these two parts were established. Two basic patterns of reproduction in higher plants were distinguished into “D-reproduction” and “I-reproduction”. The state of matter production of the mother plant determined an initial size of the daughter plant in theD-reproduction, while, in theI-reproduction, it did not determine the initial size of the daughter, but determined the number of propagules. The model of each reproduction pattern was also constructed. A formula determining the initial size of a plant in a given generation was constructed as the model of theD-reproduction. The model for theI-reproduction described the number of propagules produced in a given generation. Some aspects of the plant life, e.g. the optimum reproductive index, the switch-over time from the vegetative to the reproductive growth phase, the seed number, types of expansive reproduction, were theoretically analysed and discussed under these mathematical models.     

2-Guanidinoethanol increased dopamine release and 3,4-dihydroxyphenylacetic acid content,but not homovanillic acid content in the rat brain: Electroneurochemical and enzymological studies

The effects of 2-guanidinoethanol (GEt) on the release of monoamines and on the activity of their degrading enzymes were studied in order to investigate why 3,4-dihydroxyphenylacetic acid (DOPAC) increased to a much greater extent than homovanillic acid (HVA) after GEt injection into rat brain. In differential pulse voltammograms recorded using an electrochemically treated carbon fiber electrode, two distinct oxidation peaks, one at 130mV (DOPAC peak) and the other at 300 mV (5-hydroxyindoleacetic acid (5-HIAA) peak), were observed. In the hippocampus, the DOPAC peak increased markedly compared to the peak height recorded prior to the intracerebroventricular injection of GEt (6mol). Although the DOPAC peak height increased to 350% 4 hours after GEt injection, the 5-HIAA peak showed no change. In the striatum, the DOPAC peak increased to 150% 3 hours after GEt injection. Serial changes in the extracellular levels of DOPAC, HVA, and 5-HIAA were monitored in the striatum after GEt injection, using an in vivo brain micro-dialysis technique. Although the DOPAC levels strated to increase 80 minutes after GEt injection, HVA and 5-HIAA levels showed no change. On the other hand, monoamineoxidase, which metabolizes dopamine to DOPAC, was not activated and catechol-0-methyltransferase, which metabolizes DOPAC to HVA, were not inhibited by 5 mM of GEt in vitro. These data suggested that GEt increased the release of dopamine, but not of serotonin, and that GEt might restrict the DOPAC transport system.     

Determination of tin in biological samples using gaseous hydride generation-inductively coupled plasma-atomic emission spectrometry

A highly sensitive method determining for sub-microgram/gram levels of tin in biological samples is described. Tin hydride reduced by sodium borohydride and trichloroacetic acid solution was introduced into inductively coupled plasma after separation of liquid and excess hydrogen by an improved gas/liquid separator, and emission intensity was measured at a wavelength of 189.989 nm. Samples were decomposed by a nitric acid-perchloric acid mixture and analyzed after dilution by a standard addition technique. The relative standard deviation was 1.2% for a 10 ng/ml tin standard solution with a detection limit of 30 pg/ml.     

Polyatomics in zinc isotope ratio analysis of plasma samples by inductively coupled plasma-mass spectrometry and applicability of nonextracted samples for zinc kinetics

Inductively coupled plasma-mass spectrometry (ICP-MS) is a powerful tool for both quantitative multielement analyses of inorganic elements and measurement of isotope ratios (IRs). The main disadvantage of this technique is the existence of polyatomic isobaric interferences at some key masses. Zinc has been investigated for such potential interferences in serum or plasma. The Zn isotopes,⁶⁶Zn and⁶⁸Zn, have no apparent interferences, but³²S¹⁶O₂ and³²S₂ are isobaric with⁶⁴Zn. The possible effects of S and other major components of blood plasma—Na, K, Cl, P, Ca—on Zn IRs were investigated using a series of mineral solutions which simulated human plasma with respect to these elements. The mixture of all mineral elements interfered only with⁶⁴Zn (6.66 ng/mL) and⁷⁰Zn (8.51 ng/mL). Interferences to⁶⁶Zn,⁶⁷Zn, and⁶⁸Zn were minimal containing 0.90, 0.94, and 0.39 ng/mL, respectively. The copresence of Na or S shifted³⁵Cl¹⁶O₂ (atomic mass 67 coming from Cl solution) to³⁵Cl₂ which reduced the contribution to⁶⁷Zn. The hypothesis that Zn IRs obtained from plasma at various intervals after the intravenous administration of enriched⁶⁷Zn to humans would reflect those obtained after extraction of Zn was therefore tested. To compare the two pretreatment methods, “extraction” versus “nonextraction,” specimens were collected from 10 human subjects at intervals of 5 min to 24 h postinjection, and in 4 subjects from 5 min to 9 d postinjection. Two separate aliquots of plasma from each time-point were dried and digested with hydrogen peroxide, and the residue dissolved in nitric acid. One specimen was subjected to zinc extraction using ammonium diethyldithiocarbamate chelate followed by back extraction into nitric acid. The matching aliquot received no further pretreatment. The normalized IRs obtained from⁶⁷Zn/⁶⁶Zn and⁶⁷Zn/⁶⁸Zn in both the “extracted” and “nonextracted” samples agreed well(r ² = 0.976 andr ² = 0.985, respectively) compared to those from other ratios (r ² = 0.838 for⁶⁷Zn/⁶⁴Zn andr ² = 0.747 for⁶⁷Zn/⁷⁰Zn). Considering the minimum possibility of isobaric interferences in plasma samples,⁶⁷Zn/⁶⁸Zn obtained from “nonextracted” samples is sufficient for routine Zn kinetic analysis by ICP-MS.     

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2–7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S.cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G₁/S-boundary when quiescent human NB1–RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G₂ phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2–7 complexes.     

Anthocyanins in flowers of genus Rosa, sections Cinnamomeae (=Rosa), Chinenses, Gallicanae and some modern garden roses

Forty-four taxa of three sections (Cinnamomeae (=Rosa) 26, Chinenses 8 and Gallicanae 10) and eight modern garden roses in the genus Rosa were surveyed for their floral anthocyanins. Eleven anthocyanins: 3-glucosides and 3,5-diglucosides of cyanidin (Cy), pelargonidin (Pg) and peonidin (Pn), 3-rutinosides and 3-rho-coumaroylglucoside-5-glucosides of Cy and Pn, and Cy 3-sophoroside, were isolated from flowers of these taxa and identified by chemical and spectroscopic techniques. Four anthocyanins: Cy 3-rutinoside, Pn 3-rutinoside, Pn 3-rho-coumaroylglucoside-5-glucoside and Cy 3-sophoroside were found for the first time in Rosa flowers.Investigated sections of wild roses showed characteristic distribution of anthocyanins. Cy 3,5-diglucoside was the dominant anthocyanin detected in all three sections, but accumulation of Pn 3,5-diglucoside distinguished sections Cinnamomeae from other sections, and the occurrence of Cy 3-glucoside separates section Chinenses from others.Cy 3-sophoroside was detected in large amount in some taxa of section Cinnamomeae: e.g., R. moyesii and its related cultivars, and R. rugosa cv. Salmon Pink. The acylated Cy glycoside was found in all sections and also in some modern garden roses, while the acylated Pn glycoside was detected in the section Cinnamomeae, but not in sections Chinenses and Gallicanae. According to anthocyanin distribution patterns, eight groups were classified chemotaxonomically in genus Rosa.     

MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis

Atm1p, a mitochondrial half-type ATP-binding cassette (ABC) protein in Saccharomyces cerevisiae, transports a precursor of the iron-sulfur (Fe/S) cluster from mitochondria to the cytosol. We have identified a novel half-type human ABC protein, designating it MTABC3 (mammalian mitochondrial ABC protein 3). MTABC3 mRNA is ubiquitously expressed in all of the rat and human tissues examined. MTABC3 protein is shown to be present in the mitochondria, as assessed by immunoblot analysis and confocal microscopic analysis of subcellular fractions of Chinese hamster ovary cells stably expressing MTABC3. Accumulation of iron in the mitochondria, mitochondrial DNA damage, and respiratory dysfunction in the yeast ATM1 mutant strain (atm1-1 mutant cells) were almost fully reversed by expressing MTABC3 in these mutant cells. These results indicate that MTABC3 is a novel ortholog of the yeast and suggest an important role in mitochondrial function. Interestingly, the human MTABC3 gene has been mapped to chromosome 2q36, a region within the candidate locus for lethal neonatal metabolic syndrome, a disorder of the mitochondrial function associated with iron metabolism, indicating that MTABC3 is a candidate gene for this disorder.