Isolation of mouse transferrin using salting-out chromatography on Sepharose CL-6B.

A new method for the isolation of considerable quantities of mouse transferrin is described. This technique employs salting-out chromatography on Sepharose CL-6B, a new step in the preparation of plasma proteins. This step is followed by ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sephacryl S-200. The isolated mouse transferrin was shown to be pure by immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by the 465 nm/410 nm ratio of absorbances being 1.41. The molecular weight was determined to be about 77 500. The advantages of this procedure are that it is reproducible, gives a high recovery, and can be extended to a larger scale. The advantage over other protein purification techniques is its general utility, due to the fact that there is no need for species-specific antibodies. The application of this method offers a rapid purification of sufficient quantities of mouse transferrin essential for the elucidation of biological functions of this protein and investigations of its molecular structure.

Substrate-induced redox change of selenium in glutathione peroxidase studied by x-ray photoelectron spectroscopy.

Glutathione peroxidase showed an X-ray photoelectron spectroscopy signal of the Se 3d (3/2, 5/2) electrons at 54.4 eV. After the addition of the acceptor substrate H2O2, a marked shift of this signal to a value of 58.0 eV was observed. Upon subsequent treatment with the donor substrate glutathione, this chemical shift was reversed and the original signal was obtained. These data demonstrate that the enzyme-bound selenium moiety participates in the catalytic process. From the chemical shift obtained it is concluded that the enzyme shuttles between a selenol or selenol derivative in its reduced form and a seleninyl or selenonyl compound in its oxidized form.

Influence of lactoferrin on the function of human polymorphonuclear leukocytes and monocytes.

Polymorphonuclear leukocytes (PMN) exposed to highly purified human lactoferrin (from colostrum) exhibit an increased random motility (at least 2.5-fold) and are primed to produce more superoxide [12.1 +/- 1.2 nmol O2-/min/10(6) PMN preincubated with lactoferrin (0.5 mg/ml) against 6.4 +/- 2.3 with cells without lactoferrin after FMLP stimulation]. The action of lactoferrin seemed to be specific, because it could be abolished by simultaneous addition of antilactoferrin antibody. Addition of transferrin and iron salts to PMN was without effect. Between iron-poor and iron-saturated lactoferrin there was no difference in influence on PMN function except for a higher FMLP stimulated superoxide production by iron-saturated lactoferrin. Aggregation, degranulation (beta-glucuronidase, lysozyme), and bacterial killing were not influenced by lactoferrin. Incubation of monocytes and monocyte-derived macrophages with lactoferrin did not alter their motility or their superoxide production rates. Our findings indicate that PMN become more effective after exposure to lactoferrin by having a greater motility and producing superoxide at a faster rate.

Human milk oligosaccharides are resistant to enzymatic hydrolysis in the upper gastrointestinal tract.

BACKGROUND: Human milk oligosaccharides (HMOs) show a complexity and variety not found in milk of any other species. Although progress has been made in the past 3 decades with regard to identification and structural characterization of HMOs, not much is known about the physiologic functions of HMOs. OBJECTIVE: As a prerequisite for biological activity in infant metabolism, HMOs have to resist enzymatic hydrolysis in the gastrointestinal tract. To assess the extent to which selected HMOs are hydrolyzed, we carried out in vitro digestion studies using enzyme preparations of human and porcine pancreas and intestinal brush border membranes (BBMs). DESIGN: Fractions of HMOs, including structurally defined isolated oligosaccharides, were digested for up to 20 h with human pancreatic juice and BBMs prepared from human or porcine intestinal tissue samples. HMOs were incubated by using a porcine pancreatic homogenate and BBMs as enzyme sources. HMOs and digestion products were identified by mass spectrometry and anion-exchange chromatography. Additionally, free D-glucose, L-fucose, and N-acetylneuraminic acid were determined enzymatically. RESULTS: Whereas maltodextrin (control) was rapidly and completely hydrolyzed, neutral and acidic HMOs showed a profound resistance against pancreatic juice and BBM hydrolases. However, cleavage of most of the HMOs was achieved by using a pancreatic homogenate containing intracellular, including lysosomal, enzymes in addition to secreted enzymes. CONCLUSIONS: The results of this study strongly suggest that HMOs are not hydrolyzed by enzymes in the upper small intestine. Although intact HMOs may be absorbed, we postulate that a majority of HMOs reach the large intestine, where they serve as substrates for bacterial metabolism. Therefore, HMOs might be considered the soluble fiber fraction of human milk.

Matrix optimization for matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides from human milk.

Neutral and acidic oligosaccharides from human milk were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). These experiments require suitable matrices; their selection and particularly their preparation protocols must be optimized. Important criteria are sensitivity, reproducibility, tolerance against impurities and resolution over a wide mass range. For analytical investigations of these oligosaccharides, containing labile fucosylated and sialylated components, another property of a matrix becomes a significant factor, namely the influence on ion stability and the extent of (metastable) fragmentation. The experience gained with the MALDI/MS of neutral and acidic oligosaccharides is summarized taking into account different intentions of measurement and typical problems, such as impurities after enzymatic treatment. For a rapid screening of an oligosaccharide sample, superior results were obtained with a new preparation technique using 5-chloro-2-mercaptobenzothiazole (CMBT) as the first layer for 2,5-dihydroxybenzoic acid. For structural analysis by post-source decay, CMBT as the first layer for 3-aminoquinoline is a favoured preparation protocol, because extensive fragmentation is achieved. For acidic oligosaccharides, a special preparation protocol makes it possible to determine the number of sialic acids by inducing highly effective cationization. Matrix-assisted laser desorption/ionization mass spectrometry; matrices; oligosaccharides; post-source decay.

[3-13C] gamma-linolenic acid: a new probe for 13C nuclear magnetic resonance studies of arachidonic acid synthesis in the suckling rat.

Our objective was to develop a suitable probe to study metabolism of polyunsaturated fatty acids by 13C nuclear magnetic resonance (NMR) in the suckling rat pup. [3-13C] gamma-Linolenic acid was chemically synthesized, and a 20 mg (Experiment 1) or 5 mg (Experiment 2) dose was injected into the stomachs of 6-10-day-old suckling rat pups that were then killed over a 192 h (8 d) time course. 13C NMR showed that 13C in gamma-linolenate peaked in liver total lipids by 12-h post-dosing and that [5-13C]-arachidonic acid peaked in both brain and liver total lipids 48-96 h post-dosing. 13C enrichment in brain gamma-linolenic acid was not detected by NMR, but gas chromatography-combustion-isotope ratio mass spectrometry showed that its mass enrichment in brain phospholipids at 48-96 h post-dosing was 1-2% of that in brain arachidonic acid. 13C was present in liver and brain cholesterol and in perchloric acid-extractable water-soluble metabolites in the brain, liver and carcass. We conclude that low but measurable amounts of exogenous gamma-linolenic acid do access the suckling rat brain in vivo. The slow time course of [5-13C] arachidonic acid appearance in the brain suggests most of it was probably transported there after synthesis elsewhere, probably in the liver. Some carbon from gamma-linolenic acid is also incorporated into lipid products other than n-6 long-chain polyunsaturated fatty acids.

Mass spectrometric investigations of human milk oligosaccharides.

Human milk oligosaccharides (OS) have been fractionated by gel permeation chromatography (GPC) and analyzed by mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS) and electrospray ionization (ESI) ion trap/MS were used. Using a large human milk pool, the MALDI-TOF/MS analysis of high-molecular-mass GPC fractions showed that complex, multiply fucosylated and/or sialylated OS are present over a larger mass range than described previously Acidic oligosaccharides could be detected from low-retention-time GPC fractions with masses up to 4000 Da, which has not been reported before.

Detection of four human milk groups with respect to Lewis-blood-group-dependent oligosaccharides by serologic and chromatographic analysis.

Oligosaccharides from human milk samples obtained from individual donors were analyzed using high-pH anion-exchange chromatography. Three patterns of neutral oligosaccharides were detected corresponding to milk groups already described. These oligosaccharide groups correspond to the Lewis blood types Le(a-b+), Le(a+b-), and Le(a-b-). A new carbohydrate pattern was detected in a milk sample from a Le(a-b-) person in which only nonfucosylated oligosaccharides and compounds bearing alpha1,3-linked fucosyl residues were found. This finding led to the hypothesis that there exist 4 different oligosaccharide milk groups that fit well to the genetic basis of the Lewis blood group system.

Vitamin E status of low birthweight infants fed formula enriched with long-chain polyunsaturated fatty acids.

It is recommended in Europe that low birthweight infants (LBWI) who do not receive human milk (HM) should be fed formula enriched with long-chain polyunsaturated fatty acids (LCP). The question has been raised whether LCP supplementation to LBWI formula may have adverse effects on antioxidant status in the recipient infant, particularly on the major lipid-soluble antioxidant vitamin E (alpha-tocopherol, alpha-toc.). We studied alpha-toc. status in LBWI fed HM (n = 15) or formula either without (f, n = 8) or with LCP derived from egg lipids and fish oil (LCP-F, n = 9) on days 4 and 21 of life. Plasma alpha-toc. concentrations increased significantly in infants fed HM [d. 4: 4.53 (1.31), d. 21: 6.35 (2.18), mg/l, mean (SD), p < 0.01], whereas there were no changes in infants fed F of LCP-F. Plasma alpha-toc./total lipid ratios and erythrocyte membrane alpha-toc. concentrations did not change significantly between days 4 and 21 in either group. In contrast, erythrocyte membrane alpha-toc./total lipid ratios decreased significantly in infants fed LCP-F [0.42 (0.13) vs. 0.31 (0.06), p < 0.05], whereas no change occurred in the other two groups. We conclude that an LCP supplementation based on egg lipids and fish oil to formula may induce an early postnatal decrease of alpha-toc./total lipid ratios in erythrocyte membranes of LBWI. Therefore, the effects of different forms of LCP supplementation to infant formula on infantile vitamin E status should be carefully evaluated.

Pitfalls in the design and manufacture of infant formulae.

The composition of modern infant formulae is basically oriented on the "golden standard" human milk and influenced by several official regulations and recommendations (EC, ESPGAN, etc.). This article will focus on two recent improvements in the field of long-chain polyunsaturated fatty acids (LCPs) and protein hydrolysates. The addition of LCPs for preterm formulae was recommended recently by ESPGAN (the European Society for Pediatric Gastroenterology and Nutrition). Our research has focused on this problem for many years and we have found a good source of LCPs using specially prepared egg-yolk lipids. Others have used fish oils and run into the problem of growth retardation. Therefore, the possible sources of LCPs have to be discussed very critically and the alternatives will be shown. Also, new developments like the use of single-cell oils will be discussed. Second, the use of protein hydrolysates have been introduced for the so-called hypoantigenic or hypoallergenic formulae. Hypoantigenic formulae for preventive use have to be differentiated clearly from hypoallergenic formulae for treatment of proved cows' milk protein allergy. The problems of designing suitable hydrolysates that are low in antigenicity and good in taste will be outlined. The determination of the molecular weight distribution by gel chromatography will be compared critically with the newer techniques. The ELISA technique for testing the antigenicity is recommended before any in vivo evaluation. So far, the anaphylactic guinea-pig model is the most sensitive in vivo testing method. Summing up, modern infant formulae manufacture is much more dependent on modern laboratory techniques, which have to be chosen critically and must be adapted to the newest state of the art.

Diet and the essential fatty acid status of term infants.

Long-chain polyunsaturated fatty acids with 20 and 22 carbon atoms (LCPs) seem to play an important role during the rapid development of the infant brain in the late fetal and early postnatal period. These LCPs are integral constituents of biological membranes and they are involved in the regulation of functional properties like fluidity, permeability and activity of membrane-bound enzymes. Human milk contains LCPs in an amount of 0.5-3 wt% of total fatty acids, whereas commercially available infant formulae are almost free of them. Recently, several clinical trials, primarily with preterm infants, have reported that the content of LCPs in the blood and a functional parameter like visual acuity correlate with the content of LCPs in the diet. In this clinical trial we studied the effect of different diets on the fatty acid pattern of plasma and erythrocyte lipids of healthy term infants during the first 3 months of life. Breast-fed infants were compared with formula-fed babies who received a commercially available formula without LCPs or a new experimental formula enriched with LCPs that was similar to human milk. The results indicate that the introduction of milk feeding leads to marked differences in the blood lipid composition during the first months of life, independent of the feeding regimen. Secondly, the supplementation of a formula with LCPs seems to result in a blood lipid composition similar to infants fed with human milk. This supports the hypothesis that the newborn term infant has a limited desaturating capacity and depends on an exogenous supply of LCPs during the first months of life.

Higher urinary excretion of essential amino acids in preterm infants fed protein hydrolysates.

AIM: Protein hydrolysates have been introduced in preterm formulae, but it is not clear whether they are needed for the feeding of preterm infants. We designed a randomized, controlled trial to test the effects of a preterm formula with hydrolysed cow's milk proteins on short-term growth and urinary and plasma amino acids levels. METHODS: Infants with a birthweight < or = 1750 g and gestational age < or = 34 wk fed a conventional preterm infant formula (formula B) or a hydrolysed formula (formula A). Weight was measured daily; length, head circumference, mid-arm circumference and total skinfold thickness were measured weekly. Blood and urine were analysed for amino acid concentrations at start, 14 and 28 d. RESULTS: Twenty-one infants met the criteria for randomization. The daily feeding volumes were: formula A 172.8 +/- 5.6 vs formula B 170.1 +/- 2.8 ml/kg/d. Infants fed with formula A showed slower weight gain (17.4 +/- 3.4 vs 20.5 +/- 3.3 g/kg/d; p = 0.045) and lower mean change in Z-scores for weight (-0.18 +/- 0.16 vs 0.00 +/- 0.09; p = 0.009) and for head circumference (-0.06 +/- 0.13 vs 0.06 +/- 0.13; p = 0.049). After 14 d, infants receiving formula A had statistically significant higher urinary levels of essential amino acids compared to infants receiving formula B. CONCLUSION: Our results support the hypothesis of less nutritional value of hydrolysed versus conventional preterm formulae. Higher renal excretion of essential amino acids may be one of the mechanisms involved. These findings must be confirmed by further studies with larger sample sizes and protein hydrolysates with different degrees of hydrolysis.

Silver stain for proteins in ultrathin polyacrylamide gels: a sensitive precipitation technique.

A rapid and highly sensitive silver stain for visualization of proteins on ultrathin isoelectric focusing gels is described. This procedure is based on the specific interaction of silver and bromide ions in the presence of proteins and appears to involve a precipitation reaction. The technique requires only two reaction solutions, a silver nitrate and a potassium bromide solution. Silver consumption is very low because the silver nitrate solution is reusable. This procedure is well established for proteins separated by isoelectrofocusing in ultrathin gels.

High-performance liquid chromatographic determination of amino acids in protein hydrolysates and in plasma using automated pre-column derivatization with o-phthaldialdehyde/2-mercaptoethanol.

A sensitive and reproducible method for the routine determination of amino acids in plasma and in protein hydrolysates based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. The resolution of all amino acids was found to be good. The total time for analysis, including separation and reconditioning, ranged from 38 min for protein hydrolysates to 62 min for 29 physiological amino acids. The precision of hydrolysate analysis was within a relative standard deviation of 0.8-7.3% depending on the use of internal or external standards. The relative standard deviations of peak areas for physiological amino acids (standard) ranged between 1.8 and 5.6%. The relative standard deviations of retention times were less than 0.5% for all amino acids. This method can be used for routine analysis. One single column with 4-microns end-capped C18 material was found to be sufficient for 400-500 successive runs.

Lactoferrin stimulates colony stimulating factor production in vitro and in vivo.

A physiologic role for lactoferrin (Lf) has been implicated by (1) its antibacterial effect and (2) its involvement as a negative-feedback regulator for colony stimulating factor (CSF) and, therefore, granulocyte production. The isolation and purification of endotoxin-free, species-specific mouse and human Lf have enabled a study of the role of Lf both in vitro and in vivo. Injection of Salmonella typhimurium or LPS into mice resulted in a dose-dependent increase in plasma Lf. Treating normal and neutropenic mice with LPS showed that the plasma Lf level was directly related to the number of granulocytes found in the peripheral blood. The effect of neutropenia did not inhibit release of Lf. By incubating mouse bone marrow and adherent peritoneal cells with 0.1 microM mouse or human Lf in the absence or presence of the prostaglandin synthesis inhibitor, indomethacin (1.0 microM), no evidence could be obtained in support of a negative-feedback regulation of CSF. In fact, rather than an inhibition of CSF, the production of the latter was found to be stimulated from both cell types. Injection of endotoxin-free, mouse Lf (2 mg/animal) into mice at concentrations in the same order of magnitude as that found during bacterial infection, resulted in an increase in CSF production by 12 hours and prior to the increase in bone marrow granulocyte-macrophage progenitor cells (GM-CFC) at 48 hours. The results do not support a negative-feedback regulation of CSF by macrophages. Instead, they can be incorporated into a "demand signal" model for CSF production by macrophages.

An estimation of the soluble tubulin content in Tetrahymena cells of normal and of size-altered phenotype.

The amount of soluble tubulin in a temperature-sensitive (ts) size mutant of the ciliate Tetrahymena was measured in a variety of physiological conditions. For this purpose a competitive ELISA assay for tubulin was set up. The assay is based on an antiserum against Tetrahymena axonemal tubulin. Characterization of the antiserum shows its mono-specificity towards tubulin as well as its potential to recognize tubulin from a wide variety of cellular sources and organisms. After fractionation of the cells into soluble material, cold-labile and cold-resistant structures, we found very little tubulin soluble (less than 20% of the total), while most of the tubulin is polymerized, especially into cortical structures. Prolonged starvation does not alter the tubulin content. During the culture growth cycle the percentage of the soluble tubulin increases. Growing the ts mutant at high temperature to a large cell size will also increase the pool of soluble tubulin to a large extent. Only under this condition is the amount of soluble tubulin about equal to that fixed in cilia. The tubulins in the three different compartments are polymorphic and have a different metabolism. This is indicated by the much higher specific activity of soluble tubulin compared with the structurally bound material. In agreement, the half-life of the soluble tubulin is shorter than that of the cortical tubulin.

[The biosynthesis of glutathione in human erythrocytes (author's transl)]. / Glutathionbiosynthese, VII[1-3] Die biosynthese von Glutathion in Humanerythrozyten

The concentrations of glutathione precursors in human erythrocytes were investigated. 300muM glutamate, 375 muM glycine, and 10muM cysteine were found by automated amino acid analysis. The concentration of 2-aminobutyrate, the precursor of ophthalmic acid, was 15muM. The influence of the activities of endogenous or added glutamyl-cysteine synthetase and glutathione synthetase on the rate of glutathione biosynthesis was measured in membrane-free hemolysates under physiological conditions. The results show that the rate of the overall biosynthesis mainly depends on the formation of the dipeptide glutamyl-cysteine. The effect of glutathione precursor concentrations on the synthesis of the tripeptide was investigated at constant (endogenous) activities of the synthesizing enzymes. The rate was not enhanced by addition of glutamate and/or glycine unless cysteine or glutamyl-cysteine was also added. It is concluded that the concentration of cysteine limits the actual rate of the glutamyl-cysteine-synthetase reaction in vivo. No cysteine or bis(glutamyl)cystine was detected in human hemolysate; however, these disulfides were converted to glutathione. This indicates that erythrocytes have an appropriate system for their reduction, since the disulfides themselves are not substrates for the glutathione-synthesizing enzymes. Studies with intact human red cells indicate that the uptake of cysteine is the rate-determining step in the biosynthesis of glutathione.

Cytological and histochemical studies on the mechanism of the selective silver staining of nucleolus organizer regions (NORs).

A new silver staining method is presented (Ag-II staining) providing a rapid and reproducible way to selective silver staining of nucleolus organizer regions (NORs). In comparison with other techniques, such as the Ag-AS method and the Ag-I method, factors influencing silver stainability are discussed. Histochemical studies on the nature of the NOR-specific silver precipitate were performed either by employing various pretreatments or by inhibiting the participation ("blocking") of the various proteins or protein compounds in the staining reaction. The results would seem to indicate that the interactions of silver-ions with the carboxyl groups of acidic proteins which are involved in the rRNA-transcription process are mainly responsible for the selective silver staining of NORs.