Modulation by iron loading and chelation of the uptake of non-transferrin-bound iron by human liver cells.

Hepatic non-transferrin-bound Fe (NTBI) flux and its regulation were characterized by measuring the uptake of Fe from [59Fe]/nitrilotriacetate (NTA) complexes in control and Fe-loaded cultures of human hepatocellular carcinoma cells (HepG2). Exposure to ferric ammonium citrate (FAC) for 1 to 7 days resulted in a time- and dose-dependent increase in the rate of NTBI uptake. In contrast to previous studies showing a dependence of the rate of Fe uptake on extracellular Fe, this was positively correlated with total cellular Fe content. The Fe3+ chelating agents deferoxamine (DFO), 1,2-dimethyl-3-hydroxypyrid-4-one (CP 020) and 1,2-diethyl-3-hydroxypyrid-4-one (CP 094) prevented or diminished the increase in NTBI transport when present during Fe loading and reversed the stimulation in pre-loaded cells in relation to their abilities to decrease intracellular iron. Although saturation of the Fe uptake process was not achieved in control cells, kinetic modelling to include linear diffusion-controlled processes yielded estimated parameters of Km = 4.3 microM and Vmax = 2.6 fmol/micrograms protein/min for the underlying process. There was a significant increase in the apparent Vmax (31.2 fmol/micrograms protein per min) for NTBI uptake in Fe-loaded cells, suggesting that Fe loading increases the number of a rate-limiting carrier site for Fe. Km also increased to 15.2 microM, comparable to values reported when whole liver is perfused with FeSO4. We conclude that HepG2 cells possess a transferrin-independent mechanism of Fe accumulation that responds reversibly to a regulatory intracellular Fe pool.

Measurement of methylglyoxal in rat tissues by electrospray ionization mass spectrometry and liquid chromatography.

INTRODUCTION: Increased methylglyoxal formation due to insulin resistance has been implicated in the development of essential hypertension and in type-2 diabetic complications in animal models. Methylglyoxal is a highly reactive aldehyde, which binds sulfhydryl and amino groups of membrane proteins forming conjugates, advanced glycation end products (AGEs), which alter membrane function, leading to increased blood pressure and oxidative stress. We have shown elevated aldehyde conjugates in tissues of hypertensive rats which may be formed primarily from methylglyoxal. Our objective was to develop a specific method to measure methylglyoxal in rat tissues. METHOD: This method involves preparation of plasma, blood and tissue homogenates, solid phase extraction of methylglyoxal, derivitization using o-phenylenediamine, further purification of derivatized products by solid phase extraction and quantification by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). RESULTS: Methylglyoxal was highest in aorta followed by heart, liver, kidney and blood in that order in Sprague-Dawley rats. Levels of methylglyoxal in plasma were about an order of magnitude lower than that in tissues, but above the concentration used for the lowest calibration standard. DISCUSSION: We have successfully developed an ESI/LC/MS method for quantification of methylglyoxal in rat tissues. The high selectivity of this method offers an advantage over other methods based on fluorescence. This method will allow the evaluation of methylglyoxal in essential hypertension and type-2 diabetes.

An automated enzymatic method on the Roche COBAS MIRA S for monitoring phenylalanine in dried blood spots of patients with phenylketonuria.

OBJECTIVE: To describe and evaluate a phenylalanine dehydrogenase-coupled enzymatic assay for blood-spot phenylalanine (Phe) automated on the COBAS MIRA S analyzer for monitoring Phe in phenylketonuria (PKU) patients, as part of a home testing program. METHODS AND RESULTS: This method required a four-point calibration with each run and the useful range was 9.3 to 3500 micromol/L Phe. The within-run precision (CV%) was 8.8% at a mean of 77 micromol/L Phe and 5.3% at 787 micromol/L Phe. The between-run precision was 15% and 5.6% for 104 micromol/L and 748 micromol/L Phe, respectively. Blood-spot Phe determinations by this method were compared with plasma Phe determined by the Beckman system 7300 HPLC analyzer using 152 samples collected from PKU patients and 56 samples from patients without PKU. Linear regression analysis revealed the equation y = 0.933x + 14.9. The standard error of estimate (Sy.x) was 82.9 and the correlation coefficient (r) was 0.98. A positive bias, observed for the blood-spot Phe assay with specimens containing Phe concentrations below 200 micro mol/L, was not due to carryover or tyrosine. CONCLUSION: The results indicate that this method is acceptable for monitoring blood Phe levels in PKU patients.

Interference in glucose and other clinical chemistry assays by thiocyanate and cyanide in a patient treated with nitroprusside.

A 3-year-old patient treated with nitroprusside for congestive heart failure had 6.5 mmol/L thiocyanate (toxic, >1.5 mmol/L) and 110 micromol/L cyanide (toxic, >5 micromol/L) present in her blood. At this time a whole-blood glucose concentration assayed on the Nova Stat Profile 5 Plus (Stat Profile) was 25.1 mmol/L. Plasma from that specimen analyzed on a Kodak Ektachem 700 analyzer (E700) indicated 5.2 mmol/L glucose. We investigated the potential interference of dissolved thiocyanate or cyanide on glucose and other routine assays. Toxic concentrations of thiocyanate increased Stat Profile glucose values and E700 total calcium, chloride, and creatinine values. Stat Profile ionized calcium values were decreased by toxic concentrations of thiocyanate. Cyanide (100 micromol/L) decreased alanine aminotransferase activity measured on the E700. Interference with the Stat Profile glucose assay may have been caused by thiocyanate oxidation at the glucose electrode.

Serum prostate-specific antigen measured in children from birth to age 18 years.

We measured prostate-specific antigen (PSA) in serum from 94 cord- blood samples, from 44 newborns, and from 330 children up to age 18 years, using a highly sensitive "third generation" PSA assay on the IMMULITE (Diagnostic Products Corp.) analyzer. The serum was that remaining after cross-matching for blood transfusion. Most children were hospitalized for special care or surgery. We found detectable concentrations of PSA (> or = 0.003 micrograms/L) in many cord sera and in sera from both male and female neonates. PSA was more frequently detectable in cord and newborn sera from males than from females, but there was considerable overlap in values between the sexes, negating any possible usefulness of PSA for assigning male gender to newborns with ambiguous genitalia. PSA decreased to undetectable concentrations in most prepubertal males and females but became detectable around the age of puberty in males. We speculate that the presence of detectable PSA in cord and newborn sera results from androgenic stimulation of prostatic tissue in males or from stimulation of breast or other tissue by prolactin or progesterone in females.

Production of butyrylcholinesterase by Caco-2 cells: lack of relationship with triglyceride production.

Elevated levels of butyrylcholinesterase activity occur under a number of hypertriglyceridemic conditions, including diabetes and obesity. This study examines whether butyrylcholinesterase activity has a direct effect on triglyceride production, using Caco-2 cells, a human intestinal adenocarcinoma cell line. Caco-2 cells were incubated with 500 microM oleate to stimulate triglyceride production, and butyrylcholinesterase activity was measured in the cellular homogenate. Butyrylcholinesterase activity was approximately 3 x 10(-3) micromol/min per milligram protein. Although triglyceride production increased by almost five-fold after 18 h of stimulation with oleate, butyrylcholinesterase activity was not increased. Furthermore, inhibition of butyrylcholinesterase activity using 1 mM tetraisopropylpyrophosphoramide did not significantly affect triglyceride production or secretion. Human insulin (100 microU/ml) increased the production of butyrylcholinesterase without increasing triglyceride production. This demonstrates that stimulation of fatty acid production and butyrylcholinesterase activity occur by independent mechanisms and suggests that their correlation in hyperlipidemic conditions is not due to a direct relationship in production in situ.

Uptake of non-transferrin-bound iron by both reductive and nonreductive processes is modulated by intracellular iron.

Non-transferrin-bound iron (NTBI) uptake occurs in a variety of cells by a saturable, specific and temperature-sensitive process. Our previous studies indicated that NTBI uptake by cardiac myocytes and Hep G2 cells was reversibly up-regulated by iron deposition. In the present work we have characterized this up-regulation and examined its mechanism by comparing the uptake of oxidized (Fe3+) and ascorbate-reduced (Fe2+) forms of iron. Iron loading markedly enhanced the uptake of iron both in the presence and absence of ascorbate, but the increment was greater when ascorbate was absent. This up-regulation is partially inhibited by actinomycin D and cycloheximide, indicating a requirement for protein synthesis. Uptake by the iron-loaded cells was less sensitive to thiol-alkylating agents and competing metal ions, but was more sensitive to proteolysis. Iron loading causes an increase in both Km and Vmax for uptake of both Fe2+ and Fe3+, although the values differ, suggesting distinct rate-limiting steps for uptake of Fe2+ and Fe3+. Consistent with this idea, uptake of the two ions showed differential sensitivity to thiol reagents, competing metal ions and monensin. The Fe(2+)-specific chelators bathophenanthroline disulfonate and ferrozine markedly inhibited iron uptake whether ascorbate was present or not, indicating that Fe3+ uptake is dependent on reduction to the ferrous state. This requirement for reduction was independent of the iron status of the cells, demonstrating that the process of up-regulation is not due to the appearance of a new mechanism for translocation of Fe3+ without reduction. Taken together, the evidence favors a model of NTBI transport where an obligate and rate-determining reduction of Fe3+ occurs prior to or during uptake, followed by translocation through an Fe2+ carrier. The distinct translocation mechanisms of uptake in the presence and absence of ascorbate suggest that exogenous Fe2+ does not access the carrier available to the nascent ferrous ion derived from the reductase and is consistent with close coupling between the reduction and the translocation processes. In iron-loaded cells with increased rates of NTBI transport, a similar mechanism prevails.