A practically sensitive radioimmunoassay for cyclic CMP by 2'-O-acetylation

An improved method for the determination of subnanogram quantities of zinc has been devised using a tungsten filament for vaporization in a low-pressure microwave-induced helium plasma emission spectrometer. Desolvation and ashing in an air atmosphere of zinc containing samples in the presence of 1,10-phenanthroline (8 mm) and potassium chloride (3 mm) prevent fractional vaporization and yields a single, sharp emission signal. Analysis of nanogram quantities of zinc metalloenzymes contained in sample volumes of 5 μl illustrates the use of this method. The coefficient of variation for 0.14 ng of zinc in 75 ng of carboxypeptidase A is 3.5%, with a detection limit of 3 pg.     

Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress

In purified system zinc has been shown to have an antioxidant role. Its effects on the resistance of cultured cells towards oxidative stress in vitro were examined. Diploid human skin fibroblasts were grown for 21 d in culture media (RPMI 1640 containing 15% fetal calf serum) added with different zinc (Zn) concentrations (100, 125, and 150μM as Zinc chlorur ZnCl₂). In comparison, cell controls were grown in standard culture media (6.5μM Zn). The intracellular zinc levels of treated fibroblasts increased from 3- to 7-fold (2330±120 ng/mg protein in 150-μM Zn-treated cells versus 331±21 ng/mg protein in control cells). The intracellular copper increased 3- fold whereas the iron content slightly but not significantly decreased. The index of basal lipid peroxidation measured as thiobarbituric acid reactants (TBARs) of zinc-supplemented cells was lower than that of non zinc supplemented controls (0.89 μmol/g protein in 150μM Zn-treated cells versus 1.59 μmol/g protein in controls). At these high doses of zinc, fibroblasts expressed lower antioxidant metalloenzymes activities. Diminished TBARs in Zn treated cells tends to support that Zn acts protectively against free radical mediated damage. However when the cells were challenged with extracellular oxidant stresses mediated by hypoxanthine/xanthine oxidase or hydrogen peroxide (H₂O₂), an increased toxicity in Zn-supplemented cells was observed. When we applied an intracellular oxidative stress as UV-B or UV-A radiation, Zn-treated fibroblasts were more resistant than cells grown in normal medium. If Zn has shown antioxidant effect in some in vitro or in vivo systems our observations clearly demonstrate that this role is not mediated by antioxidant metalloenzymes.     

Enantioselective analysis of albendazole sulfoxide in plasma using the chiral stationary phase

We present a method for the enantioselective analysis of albendazole sulfoxide (ABZSO) in plasma for application in clinical pharmacokinetic studies. ABZSO enantiomers were separated on a 5-μm Chiralcel OB-H® column (4.6 × 150 mm) using hexane:ethanol (93:7, v/v) as the mobile phase and fluorescence detection. ABZSO was extracted with chloroform:isopropanol (8:2, v/v) from 500-μl aliquots of acidified plasma, with full drug recovery. The proposed method presented quantitation limits of 20 ng/ml for (−)ABZSO and 50 ng/ml for (+)ABZSO and was linear up to a concentration of 5,000 ng/ml of each enantiomer. Chirality 9:722–726, 1997. © 1997 Wiley-Liss, Inc.     

Association between plasma zinc concentration and zinc kinetic parameters in premenopausal women

The objective of this study was to measure relationships between plasma zinc (Zn) concentrations and Zn kinetic parameters and to measure relationships of Zn status with taste acuity, food frequency, and hair Zn in humans. The subjects were 33 premenopausal women not taking oral contraceptives and dietary supplements containing iron and Zn. Main outcomes were plasma Zn concentrations, Zn kinetic parameters based on the three-compartment mammillary model using 67Zn as a tracer, electrical taste detection thresholds, and food frequencies. Lower plasma Zn was significantly (P < 0.01) associated with smaller sizes of the central and the lesser peripheral Zn pools, faster disappearance of tracer from plasma, and higher transfer rate constants from the lesser peripheral pool to the central pool and from the central pool to the greater peripheral pool. The break points in the plasma Zn-Zn kinetics relationship were found between 9.94 and 11.5 micromol/l plasma Zn. Smaller size of the lesser peripheral pool was associated with lower frequency of beef consumption and higher frequency of bran breakfast cereal consumption. Hypozincemic women with plasma Zn <10.7 micromol/l or 700 ng/ml had decreased thresholds of electrical stimulation for gustatory nerves. Our results based on Zn kinetics support the conventional cutoff value of plasma Zn (10.7 micromol/l or 700 ng/ml) between normal and low Zn status.     

Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS) method for simultaneous stereoselective analysis of venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (ODV) enantiomers in human plasma has been developed and validated. Chiral chromatography is performed on the CHRIOBIOTIC V (5 microm, 250 mm x 4.6 mm) column with mobile phase constituted of 30 mmol/l ammonium acetate-methanol (15:85, pH 6.0) at a flow rate of 1.0 ml/min and a postcolumn splitting ratio of 3:1. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and detected using the selected ion recording (SIR) mode. Calibration curves obtained from spiked plasma were linear in the range of 5.0-400 ng/ml for S-(+)-VEN and R-(-)-VEN, 4.0-280 ng/ml for S-(+)-ODV and R-(-)-ODV, respectively, with linear correlation coefficient all above 0.999. The average extraction recoveries for all the four analytes were above 76%. The methodology recoveries were higher than 92%. The limit of detection were 1.0 ng/ml for S-(+)-VEN and R-(-)-VEN, 1.5 ng/ml for S-(+)-ODV and R-(-)-ODV, respectively. The intra- and inter-day variation coefficients were less than 9%.     

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.     

Development of a process for biodetoxification of metal cyanides from waste waters

A bacterial consortium capable of utilising metal cyanides as a source of nitrogen was used to develop a microbiological process for the detoxification of metal cyanides (viz. copper cyanide and zinc cyanide) from electroplating waste water. Optimal conditions biodegradation of both the metal-cyanide compounds were pH 7.5, temperature 35°C, inoculum size 10⁹ cells per ml and glucose or sugarcane molasses requirement of 5 mM or 0.6 ml/l, respectively. Metal precipitates obtained during metal-cyanide biodegradation were identified as metal-hydroxides. When the treatment was carried out in a 27 l rotating biological contactor (RBC) in continuous mode, the system could achieve >99.9% removal of 0.5 mM metal cyanide (ca. 52 mg/l cyanide and 30–40 mg/l copper/zinc) in 15 h with sugarcane molasses as carbon source. The RBC treated effluent was found to be safe for discharge in the environment as confirmed by chemical analysis and fish bioassay studies.     

Problems in the measurement of zinc using heparin as an anticoagulant

Although several reports imply that anticoagulants and preservatives contain zinc, the quantity of zinc in heparin, if any, has not been documented. Zinc concentration was determined by flame atomic absorption spectroscopy in varying dilutions of multiple commercially obtained samples of purified sodium heparin N = 15 (microgram Zn/1000 Units heparin). Rubber stoppers of sterile heparin vials and of blood evacuation tubes were incubated in pre-analyzed water or saline on a mechanical shaker with fluid aliquots obtained up to 27 hours and analyzed for zinc content (microgram Zn/0.1 ml). Heparin, with contact or without contact with rubber stoppers, recorded identical zinc concentrations. Zinc concentrations varied from 0.222 +/- 0.01 (mean +/- SE) to 3.49 +/- 0.005 microgram Zn/1000 Units heparin. Leaching of zinc from rubber stoppers of vacutainer tubes (N = 9) was noted only with those containing known chelators of zinc. These results indicate that zinc is present in certain lots of sodium heparin and that caution must be exercised when reporting zinc concentrations of blood samples that contain sodium heparin as the anticoagulant.     

Determination of the antidepressant agent citalopram and metabolites in plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of citalopram [1-(3-(dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile] and its two main metabolites (the methylamino and amino derivatives). The compounds were extracted from alkaline plasma with diethyl ether. The combined ether layers were evaporated after addition of 50 μl of 0.1 N HCl. The residual extracts were purified with diethyl ether and 20 μl were injected into a Spherisorb ODS 5-μm column with acetonitrile–0.6% phosphate buffer pH 3 (55:45, v/v) as the mobile phase. Using a fluorescence detector the detection limits are 1 ng/ml of plasma for citalopram and the methylamino metabolite and 0.5 ng/ml for the amino metabolite.     

Evaluation of an oviposition‐stimulating kairomone for the yellow fever mosquito,Aedes aegypti,in Recife,Brazil

A synthetic mixture of an oviposition‐stimulating kairomone for the yellow fever mosquito, Aedes aegypti, comprising of 83% tetradecanoic acid, 16% nonanoic acid and 1% tetradecanoic acid methyl ester (NTT, in short) was tested in a dengue endemic area in Recife, Brazil. Gravid female mosquitoes confined to a cage under semi‐field conditions deposited significantly higher numbers of eggs in traps baited with NTT at doses ranging from 0.6 to 600 ng/μl than in control (water) traps. When tested in homes, egg‐laying in traps baited with 60 ng NTT/μl (final concentration in trap, ≈3.33 ng/ml) and in control traps was not significantly different, but egg deposited in traps with lower dosage (6 ng NTT/μl; final concentration in trap, ≈0.33 ng/ml) was significantly higher than in control traps. In subsequent trials, the numbers of eggs laid in traps baited with 0.6 ng NTT/μl (final concentration in trap, ≈0.033 ng/ml) were not significantly different from the numbers deposited in trap loaded with 6 ng NTT/μl. Egg‐laying was significantly higher in these treatments than in control traps.     

Androgen binding protein, sperm production, semen output and LH and testosterone profiles in young postpubertal bulls

An androgen binding protein (ABP), which binds 5alpha-dihydrotestosterone with high affinity (Ka = 0.3 x 10(9) M(-1)), has been demonstrated in testicular and epididymal cytosols of 5 young post pubertal bulls (15-17 months old) of the Montbeliarde dairy breed. Simultaneously, daily sperm production (DSP), semen output and plasma LH and testosterone concentrations (from frequent samplings) were determined. ABP levels were 21 fmoles/mg protein in testis and 59, 22 and 43 fmoles/mg, respectively, in caput, corpus and cauda epididymis. Mean DSP, per gram of testis, was 16.6 x 10(6) spermatozoa, and the mean sperm output was approximately 1.5 x 10(9) spermatozoa per ejaculate. Mean LH and testosterone levels were 1.5 ng/ml and 2.1 ng/ml, respectively. One bull (882) was clearly distinguishable from the others, in showing higher ABP and testosterone levels together with a lower daily sperm production. Results of this study may (1) suggest a physiological role of ABP in sperm epididymal maturation and (2) give a new parameter in the evaluation of individual bulls testicular function.     

Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

Microassay for primidone and its metabolites phenylethylmalondiamide,phenobarbital and p-hydroxyphenobarbital in human serum,saliva, breast milk and tissues by gas chromatography—mass spectrometry using selected ion monitoring

A method for the quantitative determination of primidone and its metabolites phenobarbital, phenylethylmalondiamide (PEMA) and hydroxyphenobarbital (free and conjugated) in serum, urine, saliva, breast milk and tissue has been developed. Following the addition of the methyl analogues of primidone, phenobarbital and PEMA as internal standards and of saturated ammonium sulphate, the samples (5–100 μl) were extracted twice with ethyl acetate—benzene (20:80). The extracts were divided into two equal portions; one portion was ethylated by Greeley's method for the analysis of primidone, phenobarbital and hydroxyphenobarbital, while the other was trimethylsilylated for the analysis of primidone and PEMA. A gas chromatographic—mass spectrometric system was used for the analysis of the derivatized extracts. Linear calibration curves were obtained in the concentration range studied (between 100 ng/ml and 30 μg/ml). The recoveries of the drugs were between 80 and 93%. The relative standard deviations were between 3.2 and 5.9% (100-μl serum samples containing 1 μg/ml of the drugs). The lower detection limits were found to be between 1.4 and 3.7 ng/ml using serum samples of 100 μl.These methods have been applied to the study of the placental transfer and neonatal disposition of primidone and its metabolites in the human.     

Intestinal transport of zinc in the diabetic rat

Zinc has been implicated to play a role in the pathogenesis and management of diabetes. Since the intestinal transport of several minerals as calcium, magnesium and strontium was found to be altered in the diabetic rats, we postulated that intestinal zinc transport may be also altered in the diabetic rat. Therefore, using $\text{in}$$\text{vivo}$ single pass perfusion technique we determined lumen to mucosa flux, net absorption and the mucosa to lumen flux of zinc in the small and large intestinal segments of diabetic rats, diabetic rats treated with insulin and in control rats. Tissue distribution of transported ⁶⁵Zn into various organs and tissue concentrations of native zinc in the groups of rats studied were determined. Our results indicate that lumen to mucosa flux (μmole/h/g wet weight) was decreased in all intestinal segments of the diabetic rats compared to controls. However, the total capacity (mμmole/h/cm length) was similar. The specific activity and total capacity of net absorption of zinc was similar in all intestinal segments of the rats studied. The reverse mucosa to lumen flux was significantly decreased in all segments of diabetic rats compared to corresponding values in control rats. Tissue distribution of ⁶⁵Zn following the perfusion study showed increased retention of ⁶⁵Zn in the liver, kidney and femurs of the diabetic rats compared to controls. Serum and tissue concentration of native zinc in various organs were similar in all groups of rats studied. The mechanism(s) responsible for these findings are discussed.     

Serum zinc levels and goiter in Iranian school children

Iodine deficiency has been shown to have high prevalence in Iran despite sufficient iodine supplementation. Zinc deficiency may also contribute to the pathogenesis of endemic goiter. The aim of this study was to compare serum zinc level in Iranian school children with and without goiter. A cross-sectional study was performed among urban children aged 8-12 years in city of Kerman, Iran. A multistage proportional to size cluster sampling method was used to screen 5500 subjects out of 29,787 students. After the screening phase, serum and urine specimens of randomly selected 165 students were evaluated for serum zinc levels and urinary iodine excretion and compared in goiterous and non-goiterous children. Serum zinc level was 149.5±29.4 μg/l in goiterous children and 141.2±52 μg/l in non-goiterous children but no significant difference was found between the groups (p=0.37). But urinary iodine excretion was significantly (p<0.001) lower in goiterous children (207.5 μg/l in goiterous children and 262.5 μg/l in non-goiterous children). This study showed that serum zinc level in goiterous and non-goiterus children is not different and zinc deficiency is not a risk factor for endemic goiter in this population.     

Copper and zinc inhibit the metabolism of prostaglandin by the human uterus

Prostaglandins (PGs) have often been cited as intermediates in the action of the inert and copper-bearing intrauterine contraceptive device (IUD). Although investigations have shown an effect of copper at high (approx. 1 x 10(-4) mol/l) concentrations on PG synthesis, little consideration has been given to the possible effects of copper on PG metabolism. In this study the effect of copper and zinc ions on PG metabolism by human endometrium and myometrium has been investigated using radiolabel techniques together with gas chromatography mass spectrometry (GCMS) measurements of metabolites of PGE2. These experiments showed that concentration of 1 X 10(-5) mol/l of copper and zinc were sufficient to inhibit significantly (P less than 0.01) PGE metabolism. These levels of copper are within the physiological range of levels thought to be present in the uterine tissue and fluid of wearers of the copper-containing IUD and the inhibition of PG metabolism in these women might account for the small but significant decrease in the length of the luteal phase of their menstrual cycles.     

小鼠胚胎体外培养方法的优化及胚胎质量评估

实验采用输卵管收集获得的 2 细胞、原核受精卵和体外受精得到的胚胎。 2 细胞胚胎在所有培养浓度下均达到较高的囊胚发育率 ,而ISF和IVF受精卵在减低培养浓度后发育率显著降低 (p <0 .0 1 ) ,ISF受精卵从高密度 (1 /μl)下约 82 .5 %的发育率到低密度 (1 /1 0 0 0 μl)下 2 2 .3 %的发育率。而IVF胚胎的这两个值分别为 46 .3 %和5 .2 %。2 细胞胚胎与其他两种受精卵相比 ,每个囊胚所含的细胞数目差异显著 (p <0 .0 1 )。添加 1ng/ml(1 /1 0 μl)和 1 0ng/ml(1 /1 0 0 μl)的PAF显著增加了IVF胚胎的囊胚发育率 (p <0 .0 1 )。在胚胎密度为 1 /1 0 μl的培养密度下 ,添加 1 0ng/ml以上的IGF Ⅰ同样改善IVF受精卵的发育能力 (p <0 .0 1 )。培养液中添加EGF对囊胚的发育率没有影响。PAF和IGF Ⅰ协同作用的效果与IGF Ⅰ单独处理的结果并无差异 (p >0 .0 5) ,但高于PAF单独处理组 (p <0 .0 5)。结果表明 ,胚胎生长所需的生长因子在低密度培养条件下被稀释并影响胚胎的正常发育。PAF和IGF Ⅰ作为自分泌性胚胎营养因子在一定程度上补偿了由于低密度培养所带来的负效应。     

Anaemia, Folate, Zinc and Copper Deficiencies Among Adolescent Schoolgirls in Eastern Sudan

Anaemia is a widespread problem especially in the tropics. Among adolescent girls, it has negative consequences on growth, school performance, morbidity and reproductive performance. A cross-sectional study was conducted to investigate the prevalence of anaemia, iron, folate, zinc and copper deficiencies amongst adolescent schoolgirls in New Halfa, eastern Sudan, and to examine the relationship of these micronutrients with haemoglobin (Hb) levels. Out of 187 adolescent schoolgirls, 181 (96.8%) had anaemia (Hb?<?12 g/dl); 21% had mild anaemia (Hb 11.0–11.9 g/dl); 66.8.1% had moderate anaemia (Hb 8.0–10.9 g/dl), and 12.1% had severe anaemia (Hb?<?8 g/dl), respectively. Iron deficiency (S-ferritin?<?12 μg/l), iron deficiency anaemia (<12 m/dl and S- ferritin?<?12 μg/l) and folate deficiency (S-folate?<?3 ng/ml) were prevalent in 17.6%, 16.5% and 69% of these girls, respectively. Nine percent and 5.9% of these girls had zinc (<75 μg/ml) and copper deficiency (<75 μg/ml), respectively. Twenty-six (14%) girls had ≥2 micronutrient deficiencies. S-ferritin and zinc were significantly lower in patients with severe anaemia. Haemoglobin levels were significantly positively correlated with zinc levels (r?=?0.161, P?=?0.03) and with copper levels (r?=?0.151, P?=?0.03). Thus, interventions are required to prevent and control anaemia in this setting. Further research is needed.