Microenvironment around the essential cysteine residues in chicken liver fructose-1,6-bisphosphatase as analyzed by ESR spin labelling.

Chemical modification and electron spin resonance spectroscopy (ESR) spin-labelling techniques have been employed to investigate the local environment of the essential sulfhydryl groups of chicken liver fructose-1,6-bisphosphatase. The results demonstrate the presence of two distinct classes of sulfhydryl groups in this enzyme. The first class react preferentially with iodoacetate and its spin-labelled derivative, and this results in an increase in catalytic activity, while the second class react preferentially with N-ethylmaleimide and its spin-labelled derivative, and this leads to a decrease in catalytic activity. The ESR spectral data strongly suggest that the first class of sulfhydryl groups are located in a deep cleft of the enzyme molecule, while the second class of sulfhydryl groups are located in a shallow crevice. The environment of the second class of the sulfhydryl groups appears to undergo a significant change after the modification of the first class of sulfhydryl groups by iodoacetate.

Physiological roles of neuronal nicotinic receptor subtypes: new insights on the nicotinic modulation of neurotransmitter release, synaptic transmission and plasticity.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the mammalian central nervous system (CNS). Despite this, very little was known, until recently, about their physiological role. In the periphery, nicotinic receptors mediate vital excitatory fast synaptic cholinergic transmission at both the neuromuscular junction and ganglia. In the brain, this role has been mainly "delegated" to glutamate receptors. The very broad cholinergic innervations of most brain areas, including the cortex, have implicated this system, and brain nicotinic receptors in particular, in a unique "modulatory" role of other transmitters systems. Recent evidence confirms, on one hand, that brain nicotinic receptors have a dominant "presynaptic" modulatory function, controlling the release of both acetylcholine (auto-receptors) and other neurotransmitters (hetero-receptors). On the other hand, more experimental data support the idea that a variable component of fast synaptic transmission in the brain can also be mediated by "postynaptic" nicotinic receptors, which, in turn, can control cell excitability. A challenging goal is to identify which one of the plethora of nicotinic receptor subtypes is mediating each effect in different brain areas, and which of these receptors and functions are lost or affected in different human neuro-psychiatric disorders. Needless to say, a better understanding of the physiological role of brain nicotinic receptors will drive our quest for more selective and efficacious nicotinic receptor targeted therapeutic agents.

5-Hydroxyindole potentiates human alpha 7 nicotinic receptor-mediated responses and enhances acetylcholine-induced glutamate release in cerebellar slices.

The effects of 5-hydroxyindole (5-HI) have been investigated on human alpha 7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes and GH4 cells, on native alpha 7 nAChRs expressed by IMR-32 cells and on alpha 7 nAChR-mediated events in mossy fibre-granule cell synapses in rat cerebellar slices. In oocytes expressing alpha 7 nAChRs, 5-HI potentiated sub-maximal, 60 micro M ACh-induced ion currents in a concentration-dependent manner, the threshold effective concentration being 30 micro M. 5-HI itself did not act as an agonist on alpha 7 nAChRs. A maximum potentiation of 12 times the control was observed at 20 mM 5-HI. The effect of 1 mM 5-HI on the concentration-response curve for ACh revealed that 5-HI increased the potency as well as the efficacy of ACh on alpha 7 nAChRs. 5-HI also potentiated alpha 7-mediated increases in intracellular free calcium levels in both mammalian cells heterologously expressing human alpha 7 nAChRs and in human IMR-32 neuroblastoma cells expressing native alpha 7 nAChRs. At mossy fibre-granule cell synapses, application of 1 mM ACh induced glutamate-evoked excitatory post-synaptic currents (EPSCs). Co-application of 1 mM 5-HI with 1 mM ACh further increased the frequency of the EPSCs. The ACh-induced release, as well as the 5-HI-induced enhancement of release, were blocked by 1-10 nM methyllycaconitine or 200 nM alpha-bungarotoxin, demonstrating that both effects were mediated by presynaptic alpha 7 nAChRs. The results demonstrate that responses mediated by alpha 7 nAChRs are strongly potentiated by 5-HI.

Determination of cadmium in whole blood and urine by Zeeman atomic absorption spectroscopy.

Direct determination of cadmium in whole blood and urine can be achieved using Zeeman effect flameless atomic absorption spectroscopy. Within-run precision studies for whole blood cadmium determinations gave relative standard deviations of 11.3% and 6.3% for 0.53 micrograms/l and 3.16 micrograms/l, respectively. Within-run precision studies for the urine analyses yielded relative standard deviations of 11.3% and 5.2% for 0.62 micrograms/l and 2.48 micrograms/l, respectively. The detection limit is 0.12 micrograms/l in the diluted specimens. Thus, this methodology may be used to quantitate normal and toxic cadmium levels in whole blood and urine.

Separation of plasma carotenoids and quantitation of beta-carotene using HPLC.

A method was developed for the extraction and separation of human plasma carotenoids and quantitation of beta-carotene. Carotenoids were extracted from plasma with ethanol: hexane and separated by C18 reversed phase HPLC using spherical 3 micron packing. beta-Carotene was identified and quantitated using an external standard. The within-run precision of three different plasma pools ranged from 3.53-5.72% relative standard deviation (RSD). The between-run precision was 7.34% RSD. The method was linear to 500 micrograms/l with a statistical detection limit of 3.80 micrograms/l. Recovery of added beta-carotene was from 90.41-100.37%. This method was compared to a spectrophotometric 'total carotene' method. The mean plasma concentrations of 25 male and 25 female human volunteers for the 'total carotene' were 1,549 micrograms/l for all samples, 1,487 micrograms/l for males and 1,611 micrograms/l for females. The corresponding true beta-carotene concentrations obtained by HPLC analysis were 134.8, 115.9 and 153.7 micrograms/l, respectively. The true beta-carotene concentrations were on the average only 8.76% (8.07% for males and 9.46% for females) of the concentrations obtained by the spectrophotometric 'total carotene' method. Correlation between the methods had an r = 0.6107. The poor correlation is due to the difference in the measured components. Total carotene methods measure all solvent extractable moieties having absorbance in the 430-460 nm region, while the HPLC method quantitates true beta-carotene after chromatographic separation from other carotenoids. Reference intervals were established for plasma beta-carotene using REFVAL, an IFCC computer program for determining statistical reference intervals. The reference interval for all samples is 40 to 344 micrograms/l.

Determination of copper in whole blood, plasma and serum using Zeeman effect atomic absorption spectroscopy.

Methods are presented for the determination of copper in whole blood, plasma and serum using Zeeman effect flame and furnace atomic absorption spectroscopy. Three flame measurement modes were compared: continuous aspiration, microsampling in the peak height mode and microsampling in the peak area mode. The microsampler/peak area method was the most satisfactory. The precision for the microsampler/peak area method was as follows: the within-run relative standard deviation (RSD) was 1.84% and 1.89% for whole blood specimens with copper concentrations of 983 micrograms/l and 974 micrograms/l, respectively. The within-run RSD was 2.14, 1.66 and 0.87% for plasma specimens with concentrations of 990, 1,467 and 1,963 micrograms/l, respectively. Within-run RSD was 6.64, 2.86 and 1.15% for serum specimens with concentrations of 462, 984 and 2,056 micrograms/l, respectively. The average detection limit for the microsampler/peak area method was 10.3 micrograms/l. Concentrations could be read directly from standards prepared in human whole blood, plasma or serum pools or in a commercial control.

Determination of zinc and copper in urine using Zeeman effect flame atomic absorption spectroscopy.

Zinc and copper were determined in urine using polarized Zeeman effect flame atomic absorption spectrophotometry. For the zinc assay, urine was diluted 1/10 with deionized water. Concentrations could be determined by comparison to standards in a salt matrix or in a commercial urine control. The linearity of the assay was 350 micrograms/l, the detection limit was 1.2 micrograms/l and the within-run relative standard deviation (RSD) was 2.08%, 3.06%, 0.71% and 1.29% for specimens with zinc concentrations of 202 micrograms/l, 206 micrograms/l, 1 003 micrograms/l and 1 032 micrograms/l, respectively. The between-run RSD was 2.34% for a mean zinc concentration of 461 micrograms/l. For the copper assay, urine was aspirated directly and concentrations were determined by standard additions. The linearity of the assay was 5 000 micrograms/l, the detection limit was 4.6 micrograms/l and the within-run RSD was 24.49%, 16.10%, 4.00% and 3.19% for specimens with copper concentrations of 9.8 micrograms/l, 11.8 micrograms/l, 50.0 micrograms/l and 50.2 micrograms/l, respectively. The between-run RSD was 8.78% and 4.72% for specimens with copper concentrations of 21.1 micrograms/l and 40.3 micrograms/l, respectively.

Determination of zinc in whole blood, plasma and serum using Zeeman effect flame atomic absorption spectroscopy.

Methods are presented for the determination of zinc in whole blood, plasma and serum using Zeeman effect flame atomic absorption spectroscopy and a flame microsampling funnel. Whole blood was diluted 1/25 with 0.10 mol/l hydrochloric acid; plasma and serum were diluted 1/5 with deionized water. Concentrations could be read directly from standards prepared in human blood pools. The within-run relative standard deviation (RSD) was 0.50%, 0.82% and 0.61% for whole blood specimens with concentrations of 4 360 micrograms/l, 5 967 micrograms/l and 8 297 micrograms/l, respectively. The within-run RSD was 2.09%, 1.16% and 0.62% for plasma specimens with zinc concentrations of 442 micrograms/l, 976 micrograms/l and 1 731 micrograms/l, respectively. The within-run RSD was 1.18%, 1.22% and 1.02% for serum specimens with zinc concentrations of 492 micrograms/l, 1 023 micrograms/l and 1 533 micrograms/l, respectively. The detection limit was 3.6 micrograms/l.

Semi-automated determination of chromium in whole blood and serum by Zeeman electrothermal atomic absorption spectrophotometry.

Direct determination of normal and elevated levels of chromium in whole blood and serum can be achieved using Zeeman effect electrothermal atomic absorption spectrophotometry. Whole blood and serum levels of chromium were determined for an apparently healthy population and whole blood chromium levels for renal dialysis patients. Blood and serum specimens were diluted with distilled deionized water and Triton X-100. The analyses were performed utilizing air as the alternate gas to facilitate ashing in one of the char steps. Within-run precision studies for whole blood chromium determinations gave relative SD values of 4.75 and 4.65% for 0.358 and 0.172 microgram/l, respectively. Within-run precision studies for the serum chromium analysis yield relative SD values of 5.26 and 2.67% for 0.156 and 0.300 microgram/l, respectively. Detection limits were 0.025 and 0.018 microgram/l for whole blood and serum, respectively. The mean chromium level found in whole blood and serum specimens from apparently normal individuals were 0.371 microgram/l (n = 37) and 0.130 microgram/l (n = 19), with ranges of 0.120-0.673 and 0.058-0.388 microgram/l, respectively.

Preparation of d-(-)trans-1,2-diaminocyclohexane-N,N,N'N'-tetra-acetic acid and its use as a spectropolarimetric reagent for lead.

d-(-)-trans-1,2-diaminocyclohexane-N, N, N', N' -tetraacetic acid [d(-)DCTA] was prepared stereospecifically in good yield by the condensation of the resolved diamine, d-(-)-trans-1,2-diaminocyclohexane, with sodium chloroacetate at 16 degrees , followed by use of a strong cation-exchanger to obtain the free acid. Distinct differences were noted in the infrared spectra of the optically active acid and the racemate. The molecular rotation of d(-)DCTA showed strong dependence on the pH of the solution. D(-)DCTA was used as a stereospecific chelometric reagent in the spectropolarimetrically monitored determination of lead.

Determination of manganese in whole blood and serum.

We describe methods for determination of manganese in whole blood and serum with Zeeman-effect flameless atomic absorption spectroscopy. These analyses are performed on a twofold or fourfold dilution of the specimen in Triton X-100, 1 g/L. No predigestion or extraction procedures are required. The method of standard additions was used for quantitation. Within-run coefficients of variation for whole-blood manganese were 7.0 and 5.5% for 2.29 and 5.67 micrograms/L, respectively. For determination of serum manganese, coefficients of variation were 10.3 and 5.3% for 0.97 and 3.01 micrograms/L, respectively. Manganese detection limits for the assays were 3.0 pg. Whole-blood manganese concentrations, determined for 60 subjects, yielded a mean (+/- SD) of 9.03 (+/- 2.25) micrograms/L. Mean serum manganese concentration, determined for 20 subjects, was 1.82 (+/- 0.64) microgram/L. No correlation was found between blood manganese concentrations and age, sex, or smoking status.

Polarized Zeeman-effect flameless atomic absorption spectrometry of cadmium, copper, lead, and manganese in human kidney cortex.

We used polarized Zeeman-effect flameless atomic absorption spectroscopy to quantitatively measure cadmium, copper, lead, and manganese in a nitric acid digest of lyophilized human kidney cortex. Within-run coefficients of variation for cadmium, copper, lead, and manganese, 15.3, 177.2, 84.2, and 56.3 microgram/L, respectively, were 4.1, 6.3, 3.7, and 5.6%, respectively. Between-run coefficients of variation were 6.9, 5.5, 5.9, and 6.3%, respectively, for cadmium, copper, lead, and manganese concentrations of 135.1, 12.8, 2.72, and 3.80 microgram/g, respectively. For cadmium, copper, lead, and manganese digest concentrations (mean +/- SE) of 15.3 +/- 0.6, 41.4 +/- 2.6, 9.4 +/- 0.6, and 20.9 +/- 0.4 microgram/L, respectively, the detection limits were 5.2 microgram/L for copper, 1.2 microgram/L for both cadmium and lead, and 0.8 microgram/L for manganese. Assays were linear to 75 microgram/L for cadmium, 100 microgram/L for manganese, and 200 microgram/L for copper and lead. Average analytical recoveries for the four metals ranged between 95 and 101%. Because these metals were quantitated in the same digest of kidney cortex, the values for each digest gave a trace-metal profile for each autopsy specimen.

Transition metals in calf thymus deoxyribonucleoprotein.

Fe, Ni, Cu and Zn were found by energy-dispersive X-ray fluorescence in calf thymus deoxyribonucleoprotein. The X-ray analyses indicated the absence of Cr, Mn and Co.

Tissue analysis of plasticizer in dogs.

Acute renal failure was produced in animals and analytical procedures for DEHP estimation in urine and tissues of dogs developed. The presence of DEHP in urine samples of control and sham operated dogs indicated that significant excretion of this plasticizer by kidney occurred. Delay in elevation of DEHP serum levels in nephrectomized dogs and similar concentrations of DEHP in 72 hr and 96 hr serum samples in all 3 groups indicate possible metabolism and/or uptake of DEHP by tissues. DEHP levels in brain, heart, and lung tissues of nephrectomized dogs were higher than those in control and sham operated dogs; however, the highest concentration of DEHP was found in lung regardless of grouping. Gas chromatographic spectra of kidney and liver homogenates showed several extraneous peaks with shorter retention times than DEHP, probably representing metabolites of DEHP. Identification and quantitation of DEHP metabolites in tissues are presently under way in our laboratory.

Bis(2-ethylhexyl)phthalate concentrations in the serum of hemodialysis patients.

Bis(2-ethylhexyl) phthalate is extracted from artificial kidneys, both in vivo and in vitro. Perfusion of whole blood through arterial-,venous tubing (that supplied with the dialyzer) for 1 h in vitro yielded 3.23 mg of the compound, while similar perfusion of the tubing plus the artificial kidney yielded 6.10 mg. Its mean concentration after dialysis in patients undergoing hemodialysis was 751 microgram/liter of serum. Patients who had undergone more than 50 hemodialysis treatments showed significantly higher postdialysis concentrations than patients who had undergone fewer. Uptake of bis(2-ethylhexyl) phthalate by the blood during a dialysis session followed two patterns. One set of patients showed a maximum concentration at 3 h, the second set showed a steady increase until the end of dialysis. Bis(2-ethylhexyl) phthalate appeared to be rapidly cleared from blood, most being removed within 5 to 7 h of completion of dialysis.

Determination of plasticizer levels in serum of hemodialysis patients.

Serum DEHP levels were measured on 27 hemodialysis patients at various time intervals. The following results were obtained: 1) Predialysis levels of DEHP occurred in only one of 22 samples. 2) Postdialysis serum levels reached a mean of 751 nGm DEHP/ml serum. 3) Samples from patients with less than 50 hemodialysis treatments gave a mean of 558 nGm/ml, while those with more than 50 treatments gave a mean of 973 nGM/ml. These numbers were found to be significantly different at a 95% confidence level. 4) No difference in DEHP levels were found for males and females. 5) The appearance of DEHP during dialysis and its disappearance after dialysis were found to be biphasic. 6) No correlation was found between serum DEHP and triglyceride levels in postdialysis serum samples.

Bis(2-ethylhexyl) phthalate levels in nonuremic patients treated with extracorporeal devices.

DEHP levels after treatment were studied in 6 nonuremic psoriatic patients undergoing extracorporeal therapy. Serum DEHP levels at the end of therapy of these patients were significantly lower than that of maintenance hemodialysis patients. DEHP and/or its metabolites were excreted by the normal kidneys, although the excretion of nonmetabolized DEHP by the kidneys was minimal. There was no correlation between serum DEHP levels of these nonuremic patients and the type of artificial kidney used or the mode of therapy employed.