Zinc In Human Plasma Research Articles

Development of a spectrofluorometric method for selective and sensitive determination of zinc in human Plasma: Implications for olfactory function

The relationship between zinc levels and olfactory function is an area of active research due to the role of zinc-dependent metalloenzyme, carbonic anhydrase, in maintaining olfactory function. In this study a spectrofluorometric method was developed for determining zinc levels in the human plasma of healthy participants and patients with olfactory dysfunction. The method relies on the fluorescence property changes of 4-Methylesculetin, which interacts with zinc. In this interaction, the oxygen atom of hydroxyl groups serves as a ligand, coordinating with zinc as a Lewis acid. As a result, the formed complex exhibits significant fluorescence enhancement attributed to the rigid chelate structure, thereby enhancing quantum yield. The resulting complex displays emission peak at 520 nm, which is distinct from the emission peak of 4-Methylesculetin at 460 nm. This bathochromic shift indicates heightened excited-state energy, attributed to zinc coordination and complex formation. The reaction conditions were carefully optimized to ensure selective zinc determination in plasma samples. The developed method demonstrates linearity over a concentration range of 200–4000 ng/mL, with a lower limit of quantification of 115 ng/mL. When applied to quantify plasma zinc levels, the method revealed significantly decreased zinc levels in patients with olfactory dysfunction compared to healthy individuals. These results suggest a potential link between olfactory dysfunction and zinc levels (normal vs. patient; 1150 ± 19 ng/mL vs. 940 ± 14 ng/mL, p < 0.05). The developed method was validated following the ICH M10 guidelines and provided a feasible investigative link between zinc and olfactory function.

Enhanced Gadd45 expression and delayed G2/M progression are p53-dependent in zinc-supplemented human bronchial epithelial cells

Zinc is an essential nutrient for humans; however, this study demonstrated for the first time that an elevated zinc status, created by culturing cells at optimal plasma zinc concentration attainable by oral zinc supplementation, is cytotoxic for normal human bronchial epithelial (NHBE) cells. p53 plays a central role in the modulation of cell signal transduction in response to the stress from DNA damage, hypoxia and oncogene activation. The present study was designed to determine whether the previously reported increased Gadd45 expression and delayed G2/M cell cycle progression in zinc-supplemented NHBE cells is p53-dependent, and to decipher the mechanisms responsible for the regulation of Gadd45 expressions by p53, and elucidate the Gadd45 functions in impaired cell growth and cell cycle progression in NHBE cells. Cells were cultured for one passage in different concentrations of zinc: <0.4 micromol/L (ZD) as severe zinc-deficient; 4 micromol/L (ZN) as normal zinc level in culture medium; 16 micromol/L (ZA) as normal human plasma zinc level; and 32 micromol/L (ZS) as the high end of plasma zinc attainable by oral supplementation. Inhibition of cell growth and upregulation of p53 mRNA and protein expression were observed in ZS cells. Most importantly, ZS treatment also enhanced Gadd45 nuclear protein level and promoter activity, decreased CDK1-Cyclin B1 level and delayed G2/M cell cycle progression. These changes were normalized to those observed in ZN by treating ZS cells with Pifitherin, an inhibitor of p53 transactivation activity. Thus, our findings support the p53 dependency of the Gadd45-CDK1/Cyclin B1-G2/M cell cycle progression pathway in ZS NHBE cells.

Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 microM (ZD) as zinc deficient; 4 microM as normal zinc level (ZN) in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 microM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.

Suppression of Gadd45 Alleviates the G2/M Blockage and the Enhanced Phosphorylation of p53 and p38 in Zinc Supplemented Normal Human Bronchial Epithelial Cells

An adequate zinc status is essential for optimal cellular functions and growth. Yet, excessive zinc supplementation can be cytotoxic and can impair cell growth. Gadd45 plays a vital role as cellular stress sensor in the modulation of cell signal transduction in response to stress. The present study was designed to determine the influence of zinc status on Gadd45 expression and cell cycle progression in zinc deficient and supplemented normal human bronchial epithelial (NHBE) cells, and to decipher the molecular mechanism(s) exerted by the suppression of Gadd45 expression on cell growth and cell cycle progression in this cell type. Cells were cultured for one passage in different concentration of zinc: <0.4 muM (ZD) as severe zinc deficient; 4 muM as normal zinc level in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 muM (ZS) as the high end of plasma zinc attainable by oral supplementation. Inhibition of cell growth, upregulation of Gadd45 mRNA and protein expression, and blockage of G2/M cell cycle progression were observed in ZS cells. In contrast, little or no changes in these parameters were seen in ZD cells. The siRNA-mediated knocking down of Gadd45 was found to relieve G2/M blockage in ZS cells, which indicated that the blockage was Gadd45 dependent. Moreover, the enhanced phosphorylation of p38 and p53 (ser15) in ZS cells was normalized after suppression of Gadd45 by siRNA, implicating that the enhanced phosphorylation of these proteins was Gadd45 dependent. Thus, we demonstrated for the first time that an elevated zinc status modulated signal transduction to produce a delay at G2/M during cell cycle progression in NHBE cells.

Zinc deficiency depresses p21 gene expression: inhibition of cell cycle progression is independent of the decrease in p21 protein level in HepG2 cells

The influence of zinc status on p21 gene expression was examined in human hepatoblastoma (HepG2) cells. Cells were cultured for one passage in a basal medium depleted of zinc to induce severely zinc-deficient (ZD) cells or in basal medium supplemented with 0.4, 4.0, 16, or 32 microM zinc to represent mild zinc deficiency (ZD0.4), the amount of zinc in most normal media (ZN), the normal human plasma zinc level (zinc-adequate; ZA), or the high end of plasma zinc attainable by oral supplementation (ZS), respectively. In ZD and ZD0.4 cells, the nuclear p21 protein level, mRNA abundance, and promoter activity were reduced to 40, 70, and 65%, respectively, of ZN cells. However, p21 protein and mRNA levels, as well as p21 promoter activity, were not altered in ZA and ZS cells compared with ZN cells. Moreover, the amounts of acetylated histone-4 associated with the proximal and distal p21 promoter regions, as a measure of p21 promoter accessibility, were decreased in ZD (73 and 64%, respectively) and ZD0.4 (82 and 77%, respectively) cells compared with ZN cells (100 and 100%, respectively). Thus multiple lines of evidence indicate that the transcriptional process of p21 is downregulated by depressed zinc status in HepG2 cells. Furthermore, the transfection of 5 microg of plasmid cytomegalovirus-p21 plasmid, which constitutively expressed p21, was able to normalize the reduction in p21 protein level and cyclin D1-cdk4 complex activity but not the inhibition of cell growth and G1/S cell cycle progression in ZD cells.

Zinc deficiency suppresses p21 transcriptional activity in human hepatoblastoma HepG2 cells

We previously reported a slower G1/S cell cycle progression in zinc-deficient HepG2 cells. Since p21 is one of the major cell cycle regulators, the influence of zinc status on its expression was examined. Cells were cultured for one passage in media with different levels of zinc: < 0.1 and 0.4 μM as severe (ZD) and mild zinc-deficient (ZD0.4) media, respectively; 4.0 μM as in most cell culture media (ZN); 16 μM as in normal human plasma zinc level (ZA); and 32 μM to represent human plasma zinc level attained by oral supplementation (ZS). In both ZD and ZD0.4 cells, the p21 mRNA and nuclear protein levels were markedly repressed to about 60% and 40% of ZN cells, respectively. In contrast, they were not altered in ZA and ZS cells as compared to ZN cells. To prove the observed reduction in mRNA and protein level is caused by promoter activity and accessibility, the transient transfection assay of p21 promoter-reporter construct and chromatin immunoprecipitation (ChIP) assay, respectively, were performed. The p21 promoter activity of ZD and ZD0.4 cells was reduced to about 65% of ZN cells. While the amounts of acetylated histone 4 associated with the proximal and distal promoter regions of p21 were also depressed in ZD and ZD0.4 cells to about 73% and 82% of ZN cells, respectively. However, both promoter activity and accessibility were not altered in ZA and ZS cells as compared to ZN cells. Thus, the present data indicate that the p21 transcriptional process is depressed in zinc-deficient HepG2 cells.

Zinc status affects p53, gadd45, and c-fos expression and caspase-3 activity in human bronchial epithelial cells.

This study was designed to examine the influence of zinc depletion and supplementation on the expression of p53 gene, target genes of p53, and caspase-3 activity in normal human bronchial epithelial (NHBE) cells. A serum-free, low-zinc medium containing 0.4 micromol/l of zinc [zinc deficient (ZD)] was used to deplete cellular zinc over one passage. In addition, cells were cultured for one passage in media containing 4.0 micromol/l of zinc [zinc normal (ZN)], which represents normal culture concentrations (Clonetics); 16 micromol/l of zinc [zinc adequate (ZA)], which represents normal human plasma zinc levels; or 32 micromol/l of zinc [zinc supplemented (ZS)], which represents the high end of plasma zinc levels attainable by oral supplementation in humans. Compared with ZN cells, cellular zinc levels were 76% lower in ZD cells but 3.5-fold and 6-fold higher in ZA and ZS cells, respectively. Abundances of p53 mRNA and nuclear p53 protein were elevated in treatment groups compared with controls (ZN). For p53 mRNA abundance, the highest increase (3-fold) was observed in ZD cells. In contrast, the highest increase (17-fold) in p53 nuclear protein levels was detected in ZS cells. Moreover, gadd45 mRNA abundance was moderately elevated in ZD and ZA cells and was not altered in ZS cells compared with ZN cells. Furthermore, the only alteration in c-fos mRNA and caspase-3 activity was the twofold increase and the 25% reduction, respectively, detected in ZS compared with ZN cells. Thus p53, gadd45, and c-fos and caspase-3 activity appeared to be modulated by cellular zinc status in NHBE cells.

Validation of the determination of copper and zinc in blood plasma and urine by ICP MS with cross-flow and direct injection nebulization

The simultaneous determination of copper and zinc in human plasma and urine by inductively coupled plasma mass spectrometry (ICP MS) is discussed. The performances of a cross-flow nebulizer and a direct-injection nebulizer (DIN) were compared. Flow-injection-DIN-ICP MS analysis of clinical samples using 1–2 μl samples was optimized. Isobaric interferences were discussed and were demonstrated to be eliminated for the 65Cu and most of the Zn nuclides. The need for standard addition to compensate for signal suppression in the case of some serum samples was indicated. Results obtained by ICP MS using calibration with aqueous standard solutions were found to be in good agreement with those obtained by flame AAS for a batch of real blood plasma and urine samples. The methods developed were validated by analysis of several standard reference materials.

Isolation of a protein from human milk that enhances zinc absorption in humans

Humans absorb zinc more readily from human milk than from the milk of other species. The basis for this species specificity has not been elucidated. This report describes the isolation of a human milk protein which enhances zinc absorption. The consumption of bovine cow's milk supplemented with the human protein resulted in an elevation in human plasma zinc of 101% compared to a 50% rise following the ingestion of cow's milk alone. The identification of this biologically active protein in human milk establishes in part the basis for the species difference in zinc bioavailability.

Separation of protein-bound copper and zinc in human plasma by means of gel filtration -ion-exchange chromatography.

A procedure for the separation of protein-bound copper and zinc fractions and of potassium and magnesium in plasma, involving chromatography on columns of DEAE-Sepharose CL-6B, is described. Copper, magnesium and zinc were measured by atomic-absorption spectroscopy and potassium by flame photometry. Most plasma samples yielded one copper and two zinc fractions and these were identified by the use of protein “markers.” Most of the copper appeared to be bound to caeruloplasmin and a lesser amount to albumin, whereas zinc appeared to be bound mainly to globulins with a smaller, variable, amount bound to albumin. Magnesium and potassium appeared as single peaks eluting in approximately the same fractions, ahead of the albumin peak.

Spectrochemical determination of magnesium, chromium, nickel, copper and zinc in human plasma and red cells

Procedures are given for the collection and digestion of human plasma and red cells for the spectrochemical determination of Mg, Cr. Ni, Cu and Zn. Values found for the five metals are compared with those reported in the literature. The accuracy and precision of the method are good. The time required to prepare and analyze a sample has been reduced to less than half (14 h) required for the plasma analysis procedure recently reported by this laboratory. The red cells require about 17 h.

Spectrochemical determination of magnesium, chromium, nickel, copper and zinc in human plasma

Procedures are presented for the collection and digestion of human plasma for the spectrochemical determination of Mg, Cr, Ni, Cu and Zn. Data on the precision of the method are given. Values found for the five metals are compared with those given in the literature. The procedure for digesting plasma can be applied also to red cells and whole blood.