Calcium, phosphorus and magnesium balance: FM 85 fortification of human milk does not meet mineral needs of extremely low birthweight infants.

OBJECTIVE: Extremely low birthweight (<1000 g) infants are growing rapidly and their nutritional requirements for calcium, phosphorus, magnesium are high. DESIGN: Prospective, mineral balance. SETTING: The study was carried out at the Department of Neonatology, Virchow-Hospital, Charité Berlin and the Department of Molecular Trace Element Research, Hahn-Meitner-Institute Berlin. SUBJECTS: Nineteen infants <1000 g birthweight were admitted, nine infants dropped out and 10 infants (birthweight 730-995 g), fed fortified human milk were included. INTERVENTION: We collected infant's urine and feces for 72 h, a sample of human milk and infant's blood at 7 and 12 weeks of age. Elements were measured by inductively coupled plasma atomic emission spectrophotometry. RESULTS: Mean (s.d.) mineral concentration in milk was low especially at 12 weeks: calcium 9.88 (+/-3.58) mmol/l, phosphorus 7.02 (+/-3.81) mmol/l, magnesium 1.59 (+/-0.54) mmol/l. Calcium retention was minimal or negative during the study, whereas phosphorus and magnesium balances were positive. Caffeine and diuretics increased mineral excretion. Serum alkaline phosphatase was mostly <800 U/l, and 162 U/l in one infant with zinc deficiency at 12 weeks. Alkaline phosphatase correlated with absorption and retention of phosphorus, and with longitudinal growth. CONCLUSIONS: Infants <1000 g have high nutritional needs for calcium, phosphorus and magnesium, which are not met by a human milk fortifier widely used in Europe. Controlled trials are needed to assess requirements, duration and risks of mineral supplementation.

Nutritional zinc balance in extremely low-birth-weight infants.

BACKGROUND: Zinc is important for metabolism, cell growth, immunity, and defense against oxygen radicals. Extremely low-birth-weight (< 1000 g) infants have higher nutritional needs, but information on zinc is scarce. The authors performed nutritional balances in 10 infants with birth weights of 500 to 999 g and who were fed with fortified human milk. METHODS: The authors collected infant feces, urine, and blood and human milk samples during 72 hours at 7 and 12 weeks of age. Zinc concentration was measured by inductively coupled plasma-mass spectrophotometry, atomic emission spectrophotometry, and instrumental neutron activation analysis. RESULTS: Mean (SD) intake via human milk was 379 (+/- 373) microg x kg(-1) x d(-1) during both balances. Urinary excretion was high at 7 weeks of age, decreased to half at 12 week, and was negatively correlated (P < 0.01) with weight gain. Mean absorption was slightly positive at 7 weeks of age but zero or negative in most infants at 12 weeks of age. Retention was negative in all infants at both observation periods, except in one infant during the second balance. Clinical zinc deficiency developed in one infant at 12 weeks of age. CONCLUSIONS: Zinc balances in extremely low-birth-weight infants are highly variable and usually negative. Controlled trials are needed to assess need for and benefits and risks of zinc supplementation.

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry.

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.

Quantification of the metal distribution in metallothioneins of the human liver by HPLC coupled with ICP-AES.

Fractions containing metallothioneins (MT's), extracted from the liver cytosol of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for differences in the trace-element binding pattern. After the extraction of supernatants from the tissue samples, membrane ultrafiltration of the cytosolic solution was carried out to separate all high-molecular proteins with molecular weights >100 kDa. This procedure retains the metal content of the MT's in its initial form, in contrast to the often-used heat treatment of samples, which changes the copper distribution significantly. The MT's themself were isolated using size exclusion and anion exchange chromatography. Their metal content was determined simultaneously on-line by combination with an ICP-AES as element detector. Calibration of the procedure was performed by means of a column by-pass-injection of elemental standards into the separation system. The MT content in the samples was calculated using the determined metal concentrations and the generally accepted metal/protein ratios for Cu (12:1), Cd (7:1) and Zn (7:1). These values were compared with values resulting from a 109Cd-saturation-assay. When various liver samples of different pathogenesis were compared, the highest level of Cu-MT was found in primary biliary cirrhosis.

In vitro abrasion using an air-powder polishing device and its quantification by radiotracer measurement.

OBJECTIVES: This study was conducted to examine the assessment of abrasion of two different materials after neutron activation. METHODS: A fissure sealant material (Fissurit F/VOCO with a compressive strength of 235 MPa) and a glass ionomer cement (Aqua Ionofil/VOCO with a compressive strength of 170 MPa) were activated by irradiation with neutrons. Subsequent measurements of the full-energy-peak (FEP) (1368.55 keV of 24Na) were made of the sample materials before and after abrasion via exposure to the air-powder polishing to accurately describe substance loss. RESULTS: Abrasion varied more than three-fold between the two materials. SIGNIFICANCE: Neutron activation and radiotracer measurement allows the quantification of abrasion effects in different materials. In comparison with other current methods its use may allow a superior measurement accuracy and precision in determining the abraded mass.

The TSH-dependent variation of the essential elements iodine, selenium and zinc within human thyroid tissues.

Instrumental Neutron Activation Analysis was used in order to measure iodine, selenium and zinc concentration in thyroid samples. A pair of samples of normal and nodular tissue were collected from the thyroid gland from 72 patients selected on the basis of pathological criteria (44 cases of multinodular goiter, 12 of chronic lymphocytic thyroiditis (CLT), 6 of thyroid adenoma (TA) and 12 of thyroid cancer (TC)). The check for tissue homogeneity and sampling error was performed by means of the coefficient of variation (CV%) of the elements in replicate samples of normal and altered tissues. High CV% values (> 15%) for iodine reflected a functional variability in thyroid follicles, while low CV% values (< 10%) for selenium and zinc indicated that the composition of selected tissues was rather homogeneous. The variation of the element's concentration was compared in normal and altered tissues. The mean element concentrations had values close to those already reported in the literature; furthermore, our patients had marginal iodine and selenium deficiency. Both normal and nodular tissues in CLT showed statistically significant lower zinc values as compared with the other thyroid diseases. To evaluate the thyroid function, thyroid stimulating hormone (TSH) and thyroxine (T4) levels were measured in the serum of patients. Two arbitrary serum-TSH threshold levels (TSH < 1.0 and > 4.0 mU/L) were introduced in order to classify, respectively, hyperthyroidism and hypothyroidism, as well as euthyroid conditions (1.0 < TSH < 4.0 mU/L), and each patient was assigned to one of these groups. The influence of TSH in the variation of the concentration of iodine, selenium and zinc in normal and altered human thyroid tissues was significant.

Speciation as an analytical aid in trace element research in infant nutrition.

The aim of this work was twofold: to study the binding pattern of trace elements in formulas as compared with breast milk and the relationship between trace elements in breast milk and in maternal dietary intake. To investigate the binding form of trace elements in these nutritive fluids, methods for protein separation were combined with methods for trace element determination in the eluted fractions. HPLC and ICP-AES or ICP-MS were coupled on-line for the simultaneous speciation of elements of nutritional interest, viz., Ca, K, Mg, P, S, Co, Cu, Fe, I, Mn, Mo, Se and Zn, and also the heavy metals Cd and Pb in both human mild whey and formulas. In order to minimize interactions between the labile metal protein complexes and the column material, size-exclusion chromatography was used for protein separation. The binding pattern of trace elements in formulas is significantly different from that in breast milk and depends on its main component (cow's milk or soy), its processing (hydrolysis) and the chemical form (inorganic) of the added compounds. For example, compared with breast-fed infants the iron supply of formula-fed infants is much higher (up to 20-fold); in addition, the binding forms of Fe are very different in the two fluids. This has to be evaluated with respect to interactions with other elements during intestinal uptake. The investigation of breast milk samples from different regions of the world showed comparable shapes for teh elution profiles and for Mo and Se a dependence on the regional maternal dietary intake. Speciation studies carried out on breast milk samples as a function of the selenium content showed significant changes in the zinc-binding pattern. In particular, citrate (as a zinc-binding component) was found to decrease with increasing dietary selenium intake of the mother.

Separation of metalloprotein complexes in serum by size-exclusion chromatography. Optimisation of the separation parameters retention behaviour and recovery employing radiotracers.

A suitable procedure was developed for speciation analysis of metalloprotein complexes in serum using directly coupled size-exclusion chromatography and an element-specific detector. Two column matrices used for size-exclusion chromatography (TSK G 3000 SW and Asahipak GS 520) were investigated with respect to the recovery and retention behaviour for metalloprotein complexes. Optimisation of the separation parameters (buffer type, concentration, pH) was achieved by means of metalloprotein complexes marked with radiotracers. For speciation of serum the matrix in the Asahipak GS column is more efficient. Given optimal eluent characteristics (100 mM Tris, pH 7.4) the recovery of the elements investigated (sodium, calcium, iron and zinc) was 100%. Further, the retention behaviour (retention time, ratios of the peak areas) remained unchanged for several successive separations.

[Importance and physiologic nutritional requirement of the selenium trace element within the scope of parenteral nutrition (TPN)]. / Bedeutung und ernährungsphysiologischer Bedarf des Spurenelementes Selen im Rahmen der parenteralen Ernährung (TPN).

BASIS: Selenium-responsive clinical manifestations of selenium deficiency and elucidation of the biochemical and molecular biological basis of the essentiality of selenium give evidence for the biological importance of the trace element in human nutrition. CONCLUSION: The dietary parenteral selenium requirement can be calculated on the basis of the maximal gene expression of the selenoprotein plasma glutathione peroxidase (plGPx). In total parenteral nutrition a daily requirement of 0.01 mumol/kg body weight for adults and 0.025 mumol/kg body weight for children can be seen as adequate and safe.

Maternal selenium status influences the concentration and binding pattern of zinc in human milk.

To study the health effects of high dietary maternal selenium intake breast milk, blood and toe-nails were collected from 143 women (20-24 days of lactation) living in Venezuelan states of Yaracuy and Portuguesa. Depending on the regional selenium intake level three regions were defined within the total range of 90-980 microgram per day. The samples were analyzed by means of INAA for the determination of trace elements, including selenium and zinc. The significant inverse correlation between Se and Zn in breast milk found in former studies was confirmed. Investigation of the Zn-binding pattern in milk whey was carried out by an on-line combination HPLC (SEC) for protein separation and ICP-AES for element detection. Six Zn-binding compounds including citrate were detected. A highly significant negative correlation was found between the citrate, which is the main low-molecular Zn-binding compound, and the maternal daily selenium intake. We determined that the decrease in zinc concentration is due to a decrease in the citrate level, which depends on the selenium concentration in breast milk. The selenium concentration in breast milk is, in turn, proportional to the dietary intake. In addition, significant changes in the UV- and Zn-profiles were observed in the milk whey with the highest selenium content, indicating that above a certain maternal intake level substantial changes in the composition of mammary secretory cells occur. This effect can be of interest for estimation of the safe dietary intake level of selenium.

Influence of high dietary selenium intake on the thyroid hormone level in human serum.

Effects of high dietary selenium supply (range 170-980 micrograms per day) on the metabolism of thyroid hormones were studied in serum of mothers living in seleniferous areas of Venezuela. Free thyroxine (FT4), free triiodothyronine (FT3) and human thyroid stimulating hormone (hTSH) were found to be within the normal range but a significant inverse correlation was found between the FT3 and selenium. It was hypothesized that the activity of hepatic selenoenzyme type I iodothyronine 5'-deiodinase, which catalyzes the production of T3 from T4, becomes depressed at high levels of dietary intake of selenium. The effect is discussed with respect to the safe level of dietary selenium intake, which was estimated to be below 500 micrograms per day.

Protein-bound selenium in the seeds of coco de mono (Lecythis ollaria).

High selenium concentrations (7-12 g/kg dry mass) were found in the seeds of the selenium-accumulator plant coco de mono (Lecythis ollaria). In order to obtain information on the protein-bound part of selenium in extracts of these seeds, dialysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used combined with neutron activation analysis. Extractions were carried out at pH 4.5 and 7.5. In both cases about 90% of the element was dissolved. Of the extracted selenium only 9% was shown to be firmly bound to proteins at pH 4.5 and 29% at pH 7.5. For the protein-bound selenium, concentrations of 0.7 g and 2.4 g per kg of seeds and 40 and 25 g per kg of extractable protein were determined at pH 4.5 and 7.5, respectively. By analyzing the protein fractions separated by SDS-PAGE the element was found to be present in extremely selenium-rich proteins with molecular masses below 20 kDa.

The necessity of selenium substitution in total parenteral nutrition and artificial alimentation.

For the trace element selenium, in contrast to zinc, iron, copper, chromium, manganese and iodine, there is still no clear official recommendation with regard to routine substitution in artificial nutrition. An overview of the manifestations of selenium deficiency in humans during the period 1979-1995 shows that nutritive deficiencies are exclusively TPN-induced or the result of severe malnutrition. The pathology of TPN-induced selenium deficiency and the analytic assessment of selenium status are described. Patients undergoing long-term parenteral nutrition or suffering from an increased loss of intestinal secretions have to be characterized as being especially at risk for clinical selenium deficiency. The relationship of the serum selenium kinetics in pediatric and adult patients to the depletion of body compartments during the course of short-term and prolonged TPN is discussed. Because of the importance of the selenoproteins, the regularly occurring depletion during selenium-free TPN and the borderline supply of selenium in Germany the routine substitution of selenium in TPN is strongly recommended. The pharmaceutical industry should be encouraged to develop a trace element solution that includes selenium, so that the nutritive requirement of patients on TPN can be satisfied. Adequate intravenous dosage recommendations are based on maintenance of glutathione peroxidase homeostasis. The routine supplementation dosage may not meet the selenium requirements of intensive care patients under conditions of increased metabolic demands on their anti-oxidative system.

Parenteral aluminum loading in critical care medicine. Part II: Response to aluminum load from long-term parenteral nutrition.

OBJECTIVE: Evaluation of the iatrogenic aluminum load by aluminum-contaminated nutritive infusion solutions in long-term total parenteral nutrition (TPN). PATIENTS: 16 consecutive patients (6 children, 10 adults) who had to undergo total parenteral nutrition for more than one month. Three of them were from a home parenteral nutrition program. The duration of TPN was up to 68 months. The parenteral aluminum load was calculated on the basis of the individual TPN programs. Six patients were exposed to an additional parenteral aluminum load in the course of intensive care. Comparative group: To establish a reference range, the serum aluminum concentrations were determined in 71 unloaded patients who had to undergo minor surgical procedures. ALUMINUM ANALYSIS: Strict adherence to a contamination-free sampling and processing technique. The aluminum determination was performed at the Hahn-Meitner-Institute Berlin by means of graphite-furnace atomic absorption spectrometry (GFAAS). RESULTS: The TPN-associated daily aluminum load was 3.5 +/- 0.4 micrograms/kg body weight (bw) in children and 2.2 +/- 1.8 micrograms/kg bw in adults. 59 +/- 6% of the intravenous aluminum load in children and 42 +/- 16% in adults was due to the highly contaminated small-volume calcium, inorganic phosphate, trace element and vitamin parenterals. The median serum aluminum concentration under TPN was 10.9 micrograms/l (range: 5.0-26.9 micrograms/l) and was thus 7.3 times higher than in the preoperative control group (median: 1.5 micrograms/l, 95% confidence interval: < 0.6-3.5 micrograms/l). Individual values ranged up to 36.8 micrograms/l. CONCLUSIONS: The aluminum intake of patients on parenteral nutrition in Germany is thus on occasion considerably above the ASCN/ASPEN recommendations for the limitation of intravenous aluminum loading (ASCN: American Society for Clinical Nutrition; ASPEN: American Society for Parenteral and Enteral Nutrition). The toxicological significance of parenteral aluminum loading is discussed. The results suggest that limits should be established for the Aluminum contamination of infusion solutions.

Parenteral aluminum loading in critical care medicine. Part I: Aluminum content of infusion solutions and solutions for parenteral nutrition.

BACKGROUND: For patients with disturbed aluminum (Al) excretion, a high Al intake is not without risk. As main aluminum sources infusion solutions and solutions for parenteral nutrition have been identified. This study will give current survey of aluminum loading of the above-mentioned preparations. MATERIAL AND METHODS: The aluminum loading of 139 different infusion solutions and solutions for parenteral nutrition was determined. The solutions were from the clinical pharmacy of the Klinikum Steglitz of the Free University Berlin or were bought in a public pharmacy. The aluminum content was determined by means of two different, independent analytical methods: a) graphite furnace atomic absorption spectroscopy (GFAAS) and b) inductively coupled plasma atomic emission spectroscopy (ICP-AES). The agreement of the measured values was good except for five samples, where different values were found. Mistakes due to contamination were excluded on the basis of the results of measuring standard reference materials. RESULTS: Small-volume additives of TPN (total parenteral nutrition) formulations were highly contaminated with aluminum, e.g. Ca and phosphate solutions (29-12,000 micrograms/l), vitamin C solutions (700-1,200 micrograms/l) and trace element solutions (67-6,200 micrograms/l). Furthermore about 44% of the crystalline amino acid solutions and lipid emulsions had an aluminum content of 25 to 55 micrograms/l. Low aluminum levels were found in carbohydrate solutions, NaCl and KCl solutions and in distilled water (aqua ad injectabilia). CONCLUSIONS: Many of the solutions for parenteral nutritional support have an aluminum content which exceeds, in part considerably, the suggested threshold concentration of 25 micrograms/l (0.93 mumol/l), recommended by the American Society for Clinical Nutrition (ASCN) and the American Society for Parenteral and Enteral Nutrition (ASPEN). The pharmaceutical industry should be required to check the manufacturing process for avoidable sources of contamination, and threshold values for aluminum loading by intravenously applied pharmaceuticals should be laid down in the German and European pharmacopoeia. In cases where contaminations cannot be eliminated during the manufacturing process after careful checking, the aluminum content of the infusion solution should be declared for the user.

[Determination of minerals and trace elements in selenium tea from the Enschi district, People's Republic of China, and in its infusions using inductively coupled plasma-atomic emission spectrometry, graphite furnace atomic absorption spectrometry and instrumental neutron activation analysis]. / Bestimmung der Mineralstoffe und Spurenelemente in Selen-Tee aus dem Enschi-Gebiet, VR China, und in dessen Aufguss mittels ICP-AES, GFAAS und INAA.

The Enschi district in Hubei province, Peoples Republic of China is geochemically one of the two seleniferous regions, producing both selenium (Se) black tea and the Se green tea. Three samples of green tea with different Se contents and one non-Se tea were analysed. The following mineral and trace elements were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES): K, Ca, Mg, Na, P, S, Al, Mn, Fe, Ba, Sr, Co, Ni, Cu, Zn, Mo, and Cr. Except for Mo, Co, and Cr, all other elements in infusions of the samples analysed were also measured, since their concentrations are lying over their detection limits. The Se content in the tea samples and in the infusions were measured with graphite furnace atomic absorption spectrometry (GFAAS). The accuracy of Se determination was tested by measuring untreated tea samples with instrumental neutron activation analysis (INAA). The Se content in the measured samples was 1 to 8.5 micrograms/g. In addition to Se, 17 other elements were measured in the tea samples and 14 others in the infusions. With this data the extractable part of this elements in the infusion were calculated. Up to 10% of the Se was found in a first infusion.

Detection by HPLC-ICP of metallothionein in serum of an epileptic child with valproate-associated hepatotoxicity.

A patient who developed valproate-associated hepatotoxicity had significantly lower serum levels of total protein, albumin and selenium than the controls. This study shows that with the beginning of the hepatic coma metallothionein (MT) appeared in the serum mainly in the form of Zn-thionein, which altered the Zn distribution pattern of the serum in a characteristic manner. HPLC-ICP3 was successfully applied to the simultaneous speciation of elements and characterization of MT by the use of one gel permeation column.

Selenium status of children living in seleniferous areas of Venezuela.

Selenium was measured in red blood cells, serum and hair of children and in breast milk of mothers in seleniferous areas of Venezuela by means of neutron activation analysis. Signs of selenosis were observed only in a few cases. Reduction in the rate of growth is discussed with respect to unequal local nutritional conditions during the growth period.

Aluminium loading in premature infants during intensive care as related to clinical aspects.

Serum concentration and urine excretion of aluminium were examined during intensive care in 26 premature infants receiving long-time parenteral nutrition. In addition, aluminium content was analysed in all fluids and medicaments used for intravenous therapy and oral feeding. Several fluids for parenteral nutrition and milk formulae were highly contaminated with aluminium, especially serum conserves, human albumin, calcium gluconicum, and special diets for enteral disorders. Small premature infants have a predisposition to aluminium loading during intensive care. In 22 premature infants serum aluminium levels exceeded 10 micrograms/L. There was a significantly negative correlation between serum aluminium levels and gestational ages. Altogether, infants with high aluminium levels showed significantly more complications, especially bronchopulmonary dysplasia, necrotizing enterocolitis, cholestasis, and osteopenia.