Impaired phenylalanine-tyrosine conversion in patients with iron-deficiency anemia studied by a L-(2H5)phenylalanine-loading test.

Ten patients with manifest iron deficiency and without documented relationship to phenylketonuria patients were orally loaded with 25 mg/kg of L-(2H5)phenylalanine. Before loading, the fasting phenylalanine-tyrosine plasma ratio was determined and after loading, the concentrations of labeled and nonlabeled phenylalanine and tyrosine were determined in five consecutive plasma samples. With respect to the fasting phenylalanine-tyrosine ratio and to the post-load ratios of labeled phenylalanine over labeled tyrosine, the iron-deficient patients showed data intermediate between those of normals and heterozygotes for phenylketonuria. Compared to a 100% in vivo activity of phenylalanine hydroxylase in normals and a circa 37% activity in heterozygotes for classic phenylketonuria, iron-deficient patients with an average hemoglobin of 8.6 +/- 1 g/dl showed an activity of circa 56%. After normalization of their iron status, four patients were subjected again to the L-(2H5)phenylalanine-loading test. For three of these individuals, test results shifted into the range of normal.

Metabolism of iron from (3,5,5-trimethylhexanoyl)ferrocene in rats. A dietary model for severe iron overload.

The feeding of diets enriched with (3,5,5-trimethylhexanoyl)ferrocene (TMH-ferrocene) has been shown recently to produce a severe experimental iron overload in rats and has been considered as an adequate animal model for hereditary haemochromatosis in humans. We synthesized three 59Fe-labelled ferrocene compounds with different lipophilic characters (ferrocene, TMH-ferrocene, and 1,1'-bis(3,5,5-trimethylhexanoyl)ferrocene [(TMH)2-ferrocene]) and studied the metabolism of iron from these compounds in comparison with the hydrophilic ferrous sulphate in rats with iron deficiency, and normal and increased iron stores. The bioavailability of iron from TMH-ferrocene (whole body retention, 48% from a 5 mg Fe dose) was twice as high as from ferrocene and six times higher than from (TMH)2-ferrocene and ferrous sulphate. In contrast to the well-known iron salts (ferrous sulphate), the intestinal absorption of TMH-ferrocene iron was independent from the dose (1 or 5 mg Fe) and similar in iron-deficient and iron-loaded rats, indicating that the intestinal absorption of the TMH-ferrocene is not regulated by the body iron stores. After intestinal absorption, TMH-ferrocene iron in the portal blood is transported to the liver independently from transferrin. In contrast to absorbed ferrocene, iron from TMH-ferrocene is almost completely released from the hydrocarbon moiety within the liver. Depending on the body iron stores, TMH-ferrocene iron is then incorporated preferentially into haemoglobin (iron-deficient rats) or added to the iron stores in the liver (iron-loaded rats). A transient storage of the 59Fe-label in fat tissue was observed only from oral ferrocene but not from TMH-ferrocene. Due to the outstandingly high bioavailability of TMH-ferrocene, the chronic feeding of this compound resulted in a fast and progressive iron overload in rats (liver iron: 16.9 mg Fe/g wet weight after 10 weeks of feeding a diet containing 0.5% TMH-ferrocene), and can be regarded as the best characterized and most useful animal model for severe hepatocellular iron overload in humans.

Noninvasive liver-iron quantification by computed tomography in iron-overloaded rats.

RATIONALE AND OBJECTIVES: The benefit of computed tomography (CT) for the noninvasive determination of liver-iron concentration in human iron-overload diseases is a controversy in the literature. To study the sensitivity of CT for liver-iron quantification under experimental conditions, the authors measured single- and dual-energy CT numbers in vivo in the livers of iron-overloaded rats. METHODS: Thirty-five rats were subjected to an iron-rich diet for various periods, from 1 to 20 weeks, then scanned by single- and dual-energy CT. CT absorption was correlated to liver-iron content, which was determined by wet ashing and spectrophotometry. RESULTS: Whereas a good correlation (r = 0.99 at 96 kV; r = 0.95 at 125 kV) between CT numbers and liver-iron concentration was found, CT was insensitive to low concentrations of iron. Dual-energy CT scanning results showed greater scattering in liver-iron quantification compared with single-energy CT. CONCLUSIONS: In rats, the sensitivity of single- and dual-energy CT is too low to quantify liver iron in the diagnostically most relevant region of mild liver siderosis (1-3 mg iron/g wet weight [w.wt]).

Iron absorption from hemiglobin (stable oxidation product of hemoglobin) in relation to the dose in subjects with normal and depleted iron stores.

The dose relationship of hemiglobin iron absorption has been investigated in subjects with normal and depleted iron stores and was found to fit a linear regression in a bilogarithmic presentation of hemiglobin iron dose and iron absorption. Identical regression coefficients but different intercepts of regression were estimated for subjects with normal and depleted iron stores indicating a constant proportional increase of hemiglobin iron absorption in subjects with depleted iron stores in the full dose range of 0.10-50 mg hemiglobin iron.

Oral versus intravenous L-phenylalanine loading compared by simultaneous application of L-[2H5] and L-[15N]phenylalanine.

Oral loading with 1.5 g of L-[15N]phenylalanine was performed simultaneously with an intravenous infusion of 1.5 g of L-[2H5]phenylalanine in two healthy volunteers with normal phenylalanine-hydroxylase activity. For both volunteers peak levels of oral L-[15N]phenylalanine were about 20 micrograms/ml compared to peak levels of around 50 micrograms/ml for intravenous L-[2H5]phenylalanine. Throughout the four hours following application, the plasma levels of the intravenously administered phenylalanine were higher than the plasma levels of the phenylalanine administered orally. In contrast, similar plasma levels of L-[15N]tyrosine and of L-[2H4]tyrosine formed in vivo by hydroxylation of the corresponding stable isotope labelled L-phenylalanine precursors were observed during the test, indicating that about equal fractions of both the oral and of the intravenous L-phenylalanine are converted into L-tyrosine.

Proposal for the standardization of the serum unsaturated iron binding capacity assay, and results in groups of subjects with normal iron stores and with prelatent, latent, and manifest iron deficiency.

An effort has been made to standardize the indirect iron saturation excess method for the determination of the serum unsaturated iron binding capacity (UIBC) and thus to relinquish the direct adsorption methods for the assay of the serum total iron binding capacity (TIBC) which give falsely high results due to unspecific binding of the saturating iron to serum proteins. In order to eliminate the interfering effects of hydrolytic polymerization of iron(III) on the saturation of apotransferrin in serum and on the colorimetric determination of the unbound iron excess at pH 8.3, conditions have been studied for the preparation of the iron-nitrilotriacetate-complex (Fe(NTA)2) solution at pH 8.3 with respect to its reactivity with the reductant sodium ascorbate and with the chromogen bathophenanthroline-disulfonate in photometric standards and in samples containing iron-saturated serum. The validity of the results for the UIBC thus obtained has been investigated (1) by direct spectrophotometric titration with Fe(NTA)2 of the apotransferrin in serum by measuring the absorbance of transferrin at 470 nm in 50-mm cuvettes, and of the UIBC using the modified indirect iron saturation excess assay, both of which gave the same saturation points, and (2) by the correlation of the TIBC obtained from serum iron determinations and the UIBC, with the transferrin concentration measured by the radial immunodiffusion assay. Results of UIBC determinations are presented along with serum iron concentration, TIBC, and transferrin saturation in groups of subjects with normal iron stores and prelatent, latent, and manifest iron deficiency.

Stereospecificity of phenylalanine plasma kinetics and hydroxylation in man following oral application of a stable isotope-labelled pseudo-racemic mixture of L- and D-phenylalanine.

L-[15N]Phenylalanine and D-[2H5]phenylalanine have been administered orally to two healthy adult volunteers as a pseudo-racemic mixture at a dose of 25 mg/kg each. After oral application, the plasma kinetics of phenylalanine and tyrosine have been followed by the combined use of high pressure liquid chromatography and field desorption mass spectrometry. Additional incubation with D-amino acid oxidase was used to determine the enantiomeric composition of the differently labelled species of phenylalanine and tyrosine. D-Phenylalanine plasma levels show a faster rise to higher maximum values compared to L-phenylalanine (D/L ratio at maximum 3.19, 3.26). L-Phenylalanine is efficiently hydroxylated to L-tyrosine. In contrast, conversion of D-phenylalanine to the L-form with subsequent hydroxylation to L-tyrosine was observed. From the plasma kinetics it is estimated that about 1/3 of the applied dose of 25 mg/kg of D-phenylalanine is converted to the L-isomer. Of the administered dose of L-phenylalanine only very small amounts are excreted into urine as such (0.25%, 0.8%), whereas a substantial amount of the D-phenylalanine dose is found in urine (27.4%, 38.0%).

Metabolic conversion of L-[U-14C]phenylalanine to respiratory 14CO2 in healthy subjects, phenylketonuria heterozygotes and classic phenylketonurics.

Ten healthy volunteers, 12 classic phenylketonuria (PKU) heterozygotes, and 5 classic phenylketonurics have been loaded orally with a mixture of 5 microCi of L-[U-14C]phenylalanine plus 25 mg/kg of L-[2H5]phenylalanine. For 3 h thereafter, carbon-14 activity in expired air and total carbon dioxide were measured continuously and the levels of L-phenylalanine and L-tyrosine in plasma were determined in six blood samples. After 3 h, 15.1 +/- 2.1% of the applied dose of radioactivity was recovered in the expired air of the healthy subjects, compared to 10.1 +/- 2.2% for PKU heterozygotes and 0.32 +/- 0.18% for classic phenylketonurics. The integrated activity expired provides a discrimination between normals and PKU heterozygotes with a classification error of about 13% compared to an error of about 9% based on the fasting L-phenylalanine over L-tyrosine ratio. A combination of these two parameters in a two-dimensional discriminatory analysis reduces the classification error to less than 1%. An intraindividual correlation between the absolute activity expired and the formation of L-[2H4]tyrosine formed is shown, confirming that ring hydroxylation of L-phenylalanine to L-tyrosine is mandatory in the catabolism of L-phenylalanine to carbon dioxide.

Detection of heterozygous carriers for phenylketonuria by a L-[2H5]phenylalanine stable isotope loading test.

Oral loading with 25 mg/kg of pentadeuterated L-phenylalanine has been used for the discrimination between normozygous subjects and carriers for phenylketonuria. The test provides five types of data derived from plasma Phe and Tyr concentrations on which the discrimination can be based: fasting phenylalanine/tyrosine ratios, total Phe levels, total Phe/total Tyr ratios, absolute L-[2H5]phenylalanine plasma levels, and L-[2H5]Phe/L-[2H4]Tyr ratios. Absolute L-[2H4]Tyr and total L-Tyr concentrations provide the poorest discrimination with statistical classification errors around 30%. The corresponding classification error of fasting Phe/fasting Tyr ratios was circa 13%, and both labelled Phe/labelled Tyr and total Phe/total Tyr concentration ratios gave minimal errors below 2%.

Performance parameters of the Hamburg 4 pi whole body radioactivity detector.

Large 4 pi whole body counters allow the use of very low doses of radioactivity in medical research and in routine diagnosis of patients. The electronics of the 4 pi whole body counter in Hamburg (HAMCO) have recently been updated. The detector contains 1360 I of liquid organic scintillator. Experiments combined with extensive computer simulations yielded quantitative information for whole body counters and comparable systems, (1) about physical parameters such as detection efficiency, energy resolution and background count rates which are important for practical performance, and (2) concerning the optical properties of the large volume scintillation counters employed here; these are of interest for the construction of future detectors of related types.

Liver iron quantification: studies in aqueous iron solutions, iron overloaded rats, and patients with hereditary hemochromatosis.

For the noninvasive liver iron quantification by MRI in human iron overload diseases, fundamental proton relaxation mechanisms were studied in aqueous solutions with ferritin and other iron compounds, in experimentally iron overloaded rats, and in patients with iron overload diseases. MR-relaxation rates as a function of iron concentrations in the range of 0-7.5 mg Fe/g aqueous iron solutions, 0-5.4 mg Fe/g rat liver in vivo, and 0.16-4.9 mg Fe/g human liver in vivo were determined from multi- and sets of single-spin echo sequences (1.5 T imager). As predicted by theory, transverse relaxation rates (1/T2) in aqueous iron solutions, in liver tissue of rats, and in human liver tissue increased linearly with the iron concentration. A preliminary calibration for the liver iron quantification by MRI was performed from in vivo measurements of liver 1/T2-relaxation rates and liver iron quantification by atomic absorption spectroscopy in biopsies from 13 patients. With the single spin-echo method, precise in vivo liver iron quantification in humans also above 2.0 mg Fe/g liver tissue (T2 < 15 ms) should be accomplished on any imager with shortest spin-echo time available, at least TE < 20 ms.

13C-ethanol and 13C-acetate breath tests in normal and aldehyde dehydrogenase deficient individuals.

Four normal and five aldehyde dehydrogenase (ALDH) isozyme I deficient individuals were subsequently loaded with (1-13C)ethanol and (1-13C)sodium acetate and the conversion of the label to 13CO2 was determined in expired air by isotope ratio mass spectrometry. In the 13C-acetate breath test, both groups showed virtually identical recovery of the label in expired air, namely 48.5 +/- 2.3% (mean +/- S.D.) for normal and 46.8 +/- 5.7% for deficient individuals. However, in the 13C-ethanol breath test, both the groups performed differently. On average, although a certain overlap of the single data was observed, the recovery of the label after four hours was 43.4 +/- 3.8% for the normal and 35.6 +/- 6.8% for the ALDH deficient subjects. These findings suggest a slower conversion of ethanol to carbon dioxide in aldehyde dehydrogenase deficient individuals, which may be another consequence of this deficiency besides the higher plasma acetaldehyde levels observed after ethanol loading in comparison to individuals with normal aldehyde dehydrogenase activity.

Efficacy of different hexacyanoferrates(II) in inhibiting the intestinal absorption of radiocaesium in rats.

The inhibitory effect of various oral doses of different hexacyanoferrate(II) compounds (HCF) and the influence of the time interval of HCF-administration on intestinal 134Cs-absorption was studied in rats. Optimum inhibition was obtained by administration of HCF together with or 2 min before oral 134Cs loading. Using appropriate low amounts (0.1-0.5 mg) of the different HCF compounds, the inhibitory effect increased in the sequence KZnHCF less than KCuHCF less than FeHCF less than KCoHCF = KNiHCF less than NH4FeHCF = KFeHCF. Oral administration of 5 mg (0.5 mg) of KFeHCF, together with 134CsCl loading, reduces 134Cs-absorption from 41% (control) to 0.8% (2.8%). Zinc-, copper-, cobalt, and nickel hexacyanoferrates(II), despite showing a high caesium sorption capacity in vitro, were less effective in rats and are not suited for in vivo application, also because they may produce toxic side effects. As a consequence, the orally administered colloidal-soluble iron(III) hexacyanoferrates(II) (NH4Fe[Fe(CN)6] and KFe[Fe(CN)6]) have to be considered as the most valuable countermeasure against radiocaesium absorption for humans and domestic animals in the case of a severe nuclear accident in the future. Manganese oxide, a non-hexacyanoferrate(II) compound with known in vitro caesium binding capacity, showed no inhibitory effect on radiocaesium absorption in rats.

Bioavailability of iron and cyanide from 59Fe- and 14C-labelled hexacyanoferrates(II) in rats.

"Soluble" (KFe(III)[Fe(II)(CN)6]) and "insoluble Prussian blue" (Fe(III)4[Fe(II)(CN)6]3 labelled with 59Fe either in the ferric (Fe(III)) or ferro (Fe(II)) position and 14C in the cyanide group were synthesized and administered intraperitoneally or orally to adult female rats with normal body iron stores. Following i.p. injection of KFe[Fe(CN)6], the colloidal complex is disintegrated into ferric iron and hexacyanoferrate(II) anion almost completely. About 96% of the ferric iron was retained in the body. Nearly 90% of both ferrous iron and cyanide were excreted with the urine within 7 days after i.p. injection, indicating that most of the undissociated hexacyanoferrate(II) anion ([Fe(CN)6]4-) was excreted through the kidney. Only 9% of the ferrous iron from [Fe(CN)6]4- was found mainly in carcass, liver and gut. As the 59Fe/14C-ratios in organs were found close to 1.0, the dissociation of the hexacyanoferrate(II) anion can only be small in vivo. No detectable 14CO2-activity (less than 0.01%) was monitored in the breath of rats after i.p. injection of the 14C-labelled KFe[Fe(CN)6], also indicating that no significant amounts of cyanide were released after parenteral administration. After oral administration of the soluble and insoluble Prussian blue, 0.3-0.7% of the ferric iron was absorbed and retained mainly in carcass, liver and blood. Only 0.06-0.18% of the ferrous iron was absorbed and mostly excreted with the urine (0.05-0.15%), so that only 0.01-0.03% of the oral ferrous 59Fe was retained in the body after 7-10 days. Very small fractions of 14C-label from the 14CN-group of the soluble and insoluble hexacyanoferrate(II) were observed in the exhaled air (0.04-0.08% of the oral dose). From the 14CO2-exhalation, the 14C-urine excretion and the distribution of iron in blood and organs it can be concluded that the hexacyanoferrate(II) moiety disintegrated only to a small extent in the intestinal tract after oral administration. From a dose of 36 mg hexacyanoferrate(II)/kg, an amount of free (non-complex bound) cyanide can be calculated which is in maximum two orders of magnitude below the LD100-level. Thus, the very low bioavailability of iron and cyanide from hexacyanoferrate(II) compounds after oral application is demonstrated in rats. In the case of a severe nuclear accident, appropriate doses of "soluble" and "insoluble" Prussian blue can be used as safe and effective antidote against radiocaesium contamination.

205Bi/206Bi cyclotron production from Pb-isotopes for absorption studies in humans.

Pb(p, xn) thick target excitation functions were measured in the energy range 10-38 MeV in order to optimize the production of isotopically pure radiobismuth from natPb, 206Pb, and 207Pb. Additionally, the decay of Po-isotopes from deuteron irradiation of natural bismuth (209Bi) was exploited for radiobismuth production. 205Bi was produced from 206Pb at 20 MeV with only 2% of 206Bi at 4 weeks post irradiation. Bismuth compounds as used in the treatment of peptic ulcer were labeled with 205Bi for absorption studies in animals and subjects.