Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection.

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88-96%.

New high-performance liquid chromatographic method for sensitive determination of pheomelanin in biological materials by precolumn fluorescence derivatization with naphthalene-2,3-dicarboxaldehyde.

Pheomelanin is an important type of melanin distributed in the skin, eye and hair in the mammal, which is of great social, clinical and cosmetic significance. In this study, a new HPLC method with fluorescence detection is described originally for the sensitive determination of pheomelanin in biological materials. The pheomelanin polymer is decomposed into two specific degradation products, 3-amino-4-hydroxyphenylalanine (3-AHP) and 4-amino-3- hydroxyphenylalanine (4-AHP) with hydriodic acid. Then the two AHP isomers are derivatized using a fluorescent probe naphthalene-2,3-dicarboxaldehyde in the presence of cyanide. The resulting highly stable 2-substituted 1-cyanobenz[f] isoindole derivatives were separated on a 5NH(2)-MS aminopropyl packed HPLC column with binary isocratic elution profile and detected fluorimetrically. The assay shows high sensitivity of 0.11nM (2.2fmol per injection, the lowest reported) at signal-to-noise ratio of 3 for each AHPs, good accuracy and precision (RSDs<3.1%), and linearity (range of 0.02-10microM, r>0.995). The results obtained by using fluorescence detection have been compared with other detection systems (electrochemical and UV). The sensitivity can increase from 100 to respect electrochemical detection and 30000 times respect UV detection. The method has been used for the quantitative determination of pheomelanin in various biological samples, including cell cultures from five types of melanoma cell lines of human and rat origin, hair samples of various colors, melanoma tissue and the urines from human melanoma patients and healthy subjects. This original application of HPLC-fluorescence detection represents a powerful tool for investigating pheomelanin synthesis in vitro and in vivo under physiological and pathophysiological conditions.

Nitrogen interception in floodwater of rice field in Taihu region of China.

A field experiment located in Taihu Lake Basin of China was conducted, by application of urea or a mixture of urea with manure, to elucidate the interception of nitrogen (N) export in a typical rice field through "zero-drainage water management" combined with sound irrigation, rainfall forecasting and field drying. N concentrations in floodwater rapidly declined before the first event of field drying after three split fertilizations, and subsequently tended to return to the background level. Before the first field drying, total particulate nitrogen (TPN) was the predominant N form in floodwater of plots with no N input, dissolved inorganic nitrogen (DIN) on plots that received urea only, and dissolved organic nitrogen (DON) on plots treated with the mixture of urea and manure. Thereafter TPN became the major form. No N export was found from the rice field, but total nitrogen (TN) of 15.8 kg/hm2 was remained, mainly due to soil N sorption. The results recommended the zero-drainage water management for full-scale areas for minimizing N export.

Pb uptake, accumulation, subcellular distribution in a Pb-accumulating ecotype of Sedum alfredii (Hance).

Lead concentrations in roots, stems and leaves of accumulating and non accumulating ecotypes of Sedum alfredii (Hance) were studied through a hydroponic experiment with different Pb concentrations supplied as Pb(NO(3))(2). Lead concentrations in leaves and stems of the accumulating ecotype were 4 - 9 times and 3 - 5 times those of the non-accumulating ecotype, and Pb accumulated amounts in stems and leaves of the accumulating ecotype were 4 - 9 times and 8 - 11 times higher than those of the non-accumulating ecotype, respectively. The results indicated that the accumulating ecotype had better ability to transport Pb from roots to shoots. The subcellular distributions of Pb in the root, stem and leaf tissues were studied using sucrose differential centrifugation. Approximately 50% of Pb contents was found to be associated with the cell wall fraction in stems of the accumulating ecotype and the percentage increased to 80% both in roots and leaves, no matter when plants were grown with different levels of Pb. The results indicated that the distribution of Pb on cell walls of the accumulating ecotype could mainly account for the high tolerance to Pb.

Assessing copper thresholds for phytotoxicity and potential dietary toxicity in selected vegetable crops.

Copper pollution in soils is widespread, and its accumulation in crop products could pose a risk on human health. In this paper, bioavailability of added copper (Cu) and critical Cu concentrations in a vegetable garden soil was evaluated for Chinese cabbage (Brassica chinensis L.), pakchoi (Brassica chinensis L.), and celery (Apiumg graveolens L. var. dulce DC) based on human dietary toxicity. The availability of added Cu in the soil decreased with incubation time, and had minimal change after 10-12 weeks. After incubated for 12 weeks, about 60% of added Cu was not extractable by DTPA. The same crops were also grown in sand culture to determine their responses to solution Cu. Shoot growth was significantly inhibited at Cu concentrations above 10 mg kg(-1) in the solution or above 150 mg kg(-1) (DTPA-Cu) in the soil. The sensitivity of the crops to Cu toxicity differed among the three vegetable crops. Copper concentration in shoots and edible parts varied with Cu supply levels and type of the vegetables. Negative correlations (r=-0.90-0.99**) were noted between Cu concentration in shoots and fresh matter yields, but Cu concentrations in the edible parts were positively correlated with available and total Cu in the soil (r=0.91-0.99**). The critical tissue Cu concentrations at 10% shoot DM reduction were 19.4, 5.5, 30.9 mg kg(-1) for Chinese cabbage, pakchoi, and celery, respectively. Based on the threshold of human dietary toxicity for Cu (10 mg kg(-1)), the critical concentrations of total and available Cu in the soil were 430 and 269 mg kg(-1) for pakchoi, 608 and 313 mg kg(-1) for celery, and 835 and 339 mg kg(-1) for Chinese cabbage, respectively.

Differences of cadmium absorption and accumulation in selected vegetable crops.

A pot experiment and a sandy culture experiment grown with three vegetable crops of Chinese cabbage (B. chinensis L., cv. Zao-Shu 5), winter greens (B. var. rosularis Tsen et Lee, cv. Shang-Hai-Qing) and celery (A. graveolens L. var. dulce DC., cv. Qing-Qin) were conducted, respectively. The initial soil and four incubated soils with different extractable Cd (0.15, 0.89, 1.38, 1.84 and 2.30 mg Cd/kg soil) were used for the pot experiment. Five treatments were designed (0, 0.0625, 0.125, 0.250 and 0.500 mg Cd/L) in nutrient solution in the sandy culture experiment. Each treatment in pot and sandy culture experiments was trireplicated. The objectives of the study were to examine Cd accumulation in edible parts of selected vegetable crops, its correlation with Cd concentrations in vegetable garden soil or in nutrient solution, and evaluate the criteria of Cd pollution in vegetable garden soil and in nutrient solution based on the hygienic limit of Cd in vegetables. Cadmium concentrations in edible parts of the three selected vegetable crops were as follows: 0.01-0.15 mg/kg fresh weight for Chinese cabbage, 0.02-0.17 mg/kg fresh weight for winter greens, and 0.02-0.24 mg/kg fresh weight for celery in the pot experiment, and 0.1-0.4 mg/kg fresh weight for Chinese cabbage, 0.1-1.4 mg/kg fresh weight for winter greens, and 0.05-0.5 mg/kg fresh weight for celery in the pot experiment (except no-Cd treatment). The order of the three test vegetable crops for cadmium accumulation in the edible parts was celery > winter greens > Chinese cabbage in both the pot experiment and the sandy culture experiment. Cadmium accumulation in edible parts or roots of the vegetable crops increased with increasing of cadmium concentration in the medium (soil or nutrient solution). And cadmium concentrations in edible parts of the test vegetable crops were significantly linearly related to the Cd levels in the growth media (soil and nutrient solution). Based on the regression equations established and the limit of cadmium concentration in vegetable products, the thresholds of Cd concentration in the growth medium evaluated was as follows: 0.5 mg/kg soil of extractable Cd for soil and 0.02 mg/L for nutrient solution. The high capacity for cadmium accumulation in the edible parts of different vegetable crops together with the absence of visual symptoms implies a potential danger for humans.