The reaction of citraconic anhydride with bovine alpha-crystallin lysine residues. Surface probing and dissociation-reassociation studies.

Citraconic anhydride reacts readily with alpha-crystallin's lysine residues at pH 7.4. Upon addition of 2 equivalents of citraconic anhydride per equivalent lysine, 24% of the lysine residues were modified without disrupting the native quaternary structure. Further citraconylation led to dissociation into 10 S aggregates. Complete dissociation into subunits (1.4 S) occurred after adding 100 equivalents of citraconic anhydride, resulting in 98% modification. Decitraconylation did not lead to reaggregates identical with the native ones. The unmodified and the once and twice citraconylated alpha-crystallin subunits were discerned by isoelectric focusing according to their theoretical isoelectric points. In the native alpha-crystallin aggregates, nearly all B chains and approx. 60% of the A chains were found to possess at least one surface-exposed lysine residue. No differences between the susceptibilities to citraconylation of the in vivo deamidated (A1 and B1) and the de novo synthesized (A2 and B2) subunits were found. These results support the three-layer spherical assembly model for the alpha-crystallin quaternary structure.

The effect of supplementation with an enriched drink on indices of immune function in frail elderly.

Ageing induces a change in immune responses. Besides this, impaired nutritional status is considered to have a critical influence on immune function, which may be reversed by nutritional supplementation. We evaluated the effect of an enriched drink on immune function in the elderly. 33 frail elderly subjects (aged > or = 65 years and body mass index < or = 25) received two 125 ml packages of either an enriched drink (n=20) or placebo (n=13) daily for 6 months. The enriched drink contained macro- and micronutrients. At baseline and after 6 months blood samples were drawn and PBMC's were isolated. ConA stimulated proliferation and IL-2 production of PBMC's were measured. There was a significant difference between groups in proliferation over the study period. The supplement group remained stable whereas the placebo group showed a reduction in proliferation over the 6-month period. There was no significant difference in IL-2 production between groups. Our study adds to the evidence that nutritional supplementation can affect immune function in the elderly.

Human milk nonprotein nitrogen components: changing patterns of free amino acids and urea in the course of early lactation.

Free amino acids and urea were analyzed in 78 human milk samples obtained during the first 5 wk of lactation from 10 mothers delivering at term. Significant differences (p less than 0.05) in the concentrations between colostral and mature milk were found for glutamic acid, glutamine, alanine, glycine, cystine, and phosphoethanolamine which increased, and with serine, phosphoserine, aspartic acid + asparagine, arginine, lysine, isoleucine, phenylalanine, proline, methionine, tryptophan, and beta-alanine which decreased. Some of these changes occurred within the first 5 days of lactation, so that differences between transitional and mature milk became negligible (glutamic acid, alanine, and serine, aspartic acid + asparagine, lysine, isoleucine, methionine, tryptophan, respectively). No significant differences between any of the three stages of lactation were found regarding the concentrations of total free amino acids, urea, taurine, threonine, valine, leucine, histidine, and tyrosine. Possible relevances for free amino acids, including nonprotein ones, in human milk are discussed.

Double-blind, randomized trial of a synthetic triacylglycerol in formula-fed term infants: effects on stool biochemistry, stool characteristics, and bone mineralization.

BACKGROUND: The low sn-2 palmitate content of infant formulas results in formation of fatty acid calcium soaps in the stools and reduced calcium absorption. OBJECTIVE: Our objective was to test the hypotheses that increasing the proportion of sn-2 palmitate in formula for term infants would result in greater skeletal mineral deposition and reduced stool hardness. DESIGN: Healthy term neonates were randomly assigned to receive standard formula (n = 103) or formula containing 50% sn-2 palmitate (high-sn-2 formula; n = 100) for 12 wk. One hundred twenty breast-fed infants were also studied. The main outcome measures were 1) radial (single-photon absorptiometry) and whole-body (dual-energy X-ray absorptiometry) bone mineral content (WBBMC) at 12 wk and 2) stool frequency, volume, and consistency at 6 and 12 wk. Secondary outcome measures included stool fatty acid content. RESULTS: Infants receiving high-sn-2 formula had higher WBBMC (128.1 +/- 9.7 compared with 122.7 +/- 10.1 g, adjusted for size and sex), softer stools at 6 and 12 wk, and a lower proportion of stool soap fatty acids than did infants receiving the control formula. Breast-fed infants had adjusted WBBMC values (128.3 +/- 9.1 g) similar to those of infants fed high-sn-2 formula and significantly higher than those of infants fed the control formula. CONCLUSIONS: Changing the stereoisomeric structure of palmitate in infant formula resulted in higher WBBMC, reduced stool soap fatty acids, and softer stools more like those of breast-fed infants. The greater bone mass measured could be important if it persists beyond the trial period; this merits further investigation.

Age-dependent variations in the distribution of rat lens water-soluble crystallins. Size fractionation and molecular weight determination.

In order to obtain deeper insights into the mechanisms that are responsible for the age-related changes in the eye lens, the water-soluble proteins of 6-day-old to over 3-year-old rat lenses were analyzed by high-performance gel-permeation chromatography. Using this technique eleven crystallin fractions could be discerned: HM-, alpha-, three beta H-, two beta L-, beta S- and three gamma-crystallins. The concentrations of the higher molecular weight crystallins (HM-, alpha- and beta H-crystallin) seem to increase with age while those of the lower molecular weight (beta L- and gamma-crystallin) decrease. Taking into account the gradual increase of water-insoluble protein with aging, the relative amount of alpha-crystallin decreases from an age of 0.5 year after an initial increase. Additionally, an age-dependent increase in its molecular weight was found: from 7 . 10(5) to over one million. It appears that the gamma-crystallins are directly involved in the insolubilization process, while alpha- and beta L-crystallin first take part in aggregation processes leading to HM- and beta H-crystallin aggregates. These aggregation and insolubilization processes proceed gradually with increasing age. A steep decrease in gamma-crystallin concentration in the early phase of life, which also causes the relative increase in alpha-crystallin content in this period, may originate from a decrease in biosynthesis of certain gamma-crystallins.

Symptomatology and growth in infants with cow's milk protein intolerance using two different whey-protein hydrolysate based formulas in a Primary Health Care setting.

Both growth and the course of allergic symptoms were evaluated in 79 infants with cow's milk protein intolerance, aged three months or younger, diagnosed by standard elimination/provocation and treated with a whey-hydrolysate based infant formula: Nutrilon Pept or Pepti Junior. The efficacy of both products, in terms of symptomatology and growth, was compared with each other. The products differ in fat source (Pepti Junior 50% of its fat as MCT, Nutrilon Pepti normal LCT fat blend) and the presence of lactose (Pepti Junior: lactose free; Nutrilon Pepti: 40% of its carbohydrate as lactose). The study was part of a large project that aimed at standardising the approach towards cow's milk protein intolerance in Baby Health Clinics. Nearly 50 Baby Health Clinics participated in this project. In this study, growth and symptomatology (skin, respiratory tract, gastrointestinal tract) were monitored during an intervention period of at least 10 weeks. Infants in both feeding groups showed normal growth, and in at least 80% of the infants an improvement of the overall symptomatology could be seen during the intervention period. Most profound were the decreases in prevalence and severity of eczema and infantile colic. No differences in efficacy were found in this study between the two infant formulas. It was concluded that the exclusive use of either whey hydrolysate based infant formula resulted in an improvement of allergic symptoms and in normal growth in infants diagnosed by elimination/provocation for cow's milk protein intolerance in a Primary Health Care setting.

Study of the effect of a liquid nutrition supplement on the nutritional status of psycho-geriatric nursing home patients.

OBJECTIVES: To evaluate the acceptance of a multinutrient liquid nutrition supplement in psycho-geriatric nursing home patients and the effect on weight, plasma nutrients and activities of daily life. DESIGN: Double-blind, placebo-controlled 12-week intervention study. SETTING: Two nursing homes in Boxtel, The Netherlands. SUBJECTS: Forty-two (body mass index (BMI) <23 kg/m2 for men or <25 kg/m2 for women) psycho-geriatric nursing home patients aged 60 y or over. INTERVENTIONS: Provision with a complete micronutrient-enriched liquid nutrition supplement of 125 ml and 0.6 MJ (135 kcal) or placebo twice daily during daytime between main meals. Study parameters were assessed at 0, 6 and 12 weeks. MAIN OUTCOME FOR MEASURES: Weight, Barthel index of daily activities, several plasma values (albumin, C-reactive protein (CRP), homocysteine, thiamine, thiamine diphosphate (TDF), vitamin B6, vitamin B12, folic acid, vitamin D), bowel function. RESULTS: The supplement was well accepted. Thirty-five patients completed the intervention period (16 control group; 19 supplement group). Baseline daily nutrient intake was low. A statistically significant improvement was observed for body weight (difference between groups 2.2 kg, P=0.03), and homocysteine, vitamin B1, TDF, vitamin B6, vitamin B12, folate and vitamin D in the supplement group compared to the placebo group. No significant difference was observed in the Barthel index (mean difference -0.3+/-1.1 for both groups). No difference in occurrence of diarrhoea was observed. CONCLUSIONS: The study shows that nutritional supplementation is well accepted and can improve the nutritional status of psycho-geriatric nursing home patients. SPONSORSHIP: Numico Research BV.

Effect of a nutrient-enriched drink on dietary intake and nutritional status in institutionalised elderly.

OBJECTIVE: (1) To determine whether nutritional supplementation (energy and micronutrients) in institutionalised elderly has a positive effect on dietary intake and nutritional status. (2) To investigate whether individuals tend to compensate for the energy content of the intervention product by decreasing their habitual food consumption. METHODS: A 24-week, randomised, double-blind, placebo-controlled, intervention trial in homes for the elderly (n=3), in nursing homes (n=3) and 'mixed' homes (n=3) in The Netherlands. Institutionalised elderly people (n=176) older than 60 years of age, with a body mass index < or =30 kg/m(2) and a Mini-Mental State Examination score of 10 points or higher, randomly received a nutrient-enriched drink or a placebo drink twice a day during 24 weeks in addition to their usual diet. Allocation to treatment took into account of sex, the Mini-Mental State Examination score and the plasma homocysteine level. Body weight and several nutrition-related analyses in fasting blood samples were measured in all participants. Data on dietary intake were collected in a subsample (n=66). RESULTS: A significantly favourable effect (P<0.001) of the intervention drink was observed on vitamin intake, mineral intake and vitamin status in blood (for example, homocysteine decreased from 14.7 to 9.5 micromol/l in the intervention group as compared with that in the placebo group (17.2-15.9)). The difference in change in total energy intake between the two treatment groups was 0.8 MJ/day (P=0.166). Energy intake from food decreased in both groups to the same extent (-0.5 MJ/day). Therefore, this decrease cannot be considered as compensation for the energy content of the product. CONCLUSIONS: This group of institutionalised elderly people does not compensate for the energy content of a concentrated nutritional supplement. Therefore, this supplement is effective for counteracting the development of malnutrition in this population.

Faecal SIgA secretion in infants fed on pre- or probiotic infant formula.

Secretory immunoglobulin A (SIgA) plays an important role in the defence of the gastrointestinal tract. The level of faecal SIgA antibody is associated with increased neutralization and clearance of viruses. Formula-fed infants who lack the transfer of protective maternal SIgA from breast milk may benefit from strategies to support maturation of humoral immunity and endogenous production of SIgA. We aimed at studying the effects of standard, prebiotic and probiotic infant formulas on the faecal SIgA levels. At birth, infants of whom the mother had decided not to breastfeed were allocated to one of three formula groups in a randomized, double-blind fashion. Nineteen infants received standard infant formula; 19 received prebiotic formula containing a specific mixture of 0.6 g galacto-oligosaccharides (GOS)/fructo-oligosaccharides (FOS)/100 ml formula and 19 received probiotic formula containing 6.0 x 10(9) cfu Bifidobacterium animalis/100 ml formula. Faecal samples were taken on postnatal day 5, day 10, wk 4 and every 4 wk thereafter until wk 32. SIgA in faeces was determined by an enzyme-linked immunosorbent assay. During the intervention, infants fed on prebiotic formula showed a trend towards higher faecal SIgA levels compared with the standard formula-fed infants reaching statistical significance at the age of 16 wk. In contrast, infants fed on the probiotic formula showed a highly variable faecal SIgA concentration with no statistically significant differences compared with the standard formula group. Formula-fed infants may benefit from infant formulas containing a prebiotic mixture of GOS and FOS because of the observed clear tendency to increase faecal SIgA secretion. Adding viable B. animalis strain Bb-12 to infant formula did not reveal any sign for such a trend.

Homocysteine and cognitive function in institutionalised elderly A cross-sectional analysis.

BACKGROUND: Several cross-sectional, case-control and prospective studies revealed a relation between homocysteine and cognitive function or dementia. These studies included either patient populations or healthy, community- dwelling elderly people. AIM OF THE STUDY: In this study we tested the hypothesis that homocysteine was inversely associated with cognitive function in a population of institutionalised elderly (aged >/= 60 y; n = 157). METHODS: For testing this hypothesis baseline data of a recently conducted intervention study in institutionalised elderly (median age 83 years) were used. Cognitive function was evaluated by the cognitive subscale of the Alzheimer's disease Assessment Scale (ADAS-cog). The association between fasting plasma homocysteine level and cognitive function was investigated by multiple linear regression analysis. RESULTS: In the crude model homocysteine concentration was not significantly related to ADAS-cog score (beta = 0.061; p = 0.45). Age was found to be related to ADAS-cog score (beta = 0.161; p < 0.05). Adjusting for age did however not result in a relation between homocysteine and cognitive function. CONCLUSIONS: In our study no association was found between homocysteine and cognitive function in a population of very old institutionalised subjects.

Biochemistry of maternal milk in early lactation.

The analysis of more than 550 human milk samples showed that triglycerides (accounting for 96-99 per cent of the total lipids) increased from 2 to 3.5 g/100 ml mainly during the first week postpartum and remained constant thereafter. In contrast, both cholesterol and vitamin E concentrations decreased from 35 to 20 mg/100 ml and from 1.7 to 0.30 mg/100 ml, respectively. The phospholipids remained constant (40 mg/100 ml). The fatty acid composition of total lipids showed remarkable changes. Mid-chain fatty acids (C10, C12, C14) increased, whereas the long-chain polyunsaturated fatty acids decreased. The P/S ratio of 0.32 remained constant throughout lactation. The mean protein content of human milk decreased from approximately 2 g/100 ml at day 2 to approximately 1 g/100 ml at day 36 of lactation. The content of each individual amino acid decreased likewise. However, when we expressed the amino acid values in relation to the protein value--that is as gram of amino acid per gram of protein--some decreased, some remained constant and some increased indicating a changing protein pattern. Throughout the whole period of investigation, non-protein nitrogen accounted for about 20 per cent of total nitrogen, taurine ranging at about 6 mg/100 ml. The calcium content increased from a mean value of 25 mg/100 ml at day 1 to 32 mg/100 ml at day 5 and remained constant at 30 mg/100 ml up to day 36. The mean phosphorus content increased from 10 mg/100 ml at day 1 to 17 mg/100 ml at day 8 and then continuously decreased to 13 mg/100 ml at day 36.

The quaternary structure of bovine alpha-crystallin. Size and charge microheterogeneity: more than 1000 different hybrids?

Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5--16.0 nm (maximum at 14.0--15.0 nm) for native alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native alpha-crystallin. No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2, A1, A1/2-151, B2 and B1 conceivable.

Protein patterns obtained from support-bound gels by combining the images at different stages of destaining.

A more informative method for the visualization of proteins on thin-layer gels, based on combining the images of the gel at different stages of destaining, is presented. It is useful whenever important information seems to be lost after prolonged destaining. The method, which makes it unnecessary to run different loads in different channels, has been developed utilizing isoelectric focusing on polyester film-bound agarose gels. The strongly destained gel is superimposed on a negative image of the same gel made at an earlier phase of destaining, thus showing white spots on a gray background for minor components and dark bands in a white field surrounded by the grey background for the abundant ones. In general, the method may be applied to gel images obtained by different staining procedures.

The quaternary structure of bovine alpha-crystallin. Effects of variation in alkaline pH, ionic strength, temperature and calcium ion concentration.

The stability of the native quaternary structure of bovine alpha-crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7--11), ionic strength (0.01--0.5), temperature (6--60 degrees C) and calcium ion concentration (0 and 10 mM). Three successive transitions are distinguished at 20 degrees C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of 'alkali-modified alpha-crystallin', which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali-modified alpha-crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3. Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37 degrees C with formation of 'temperature-modified alpha-crystallin', which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37 degrees C a reversal of this transition or aggregation is indicated. These findings, together with previous structural data on microheterogeneity, reassociation from urea, and aging of alpha-crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three-layer model for the quaternary structure.

The quaternary structure of bovine alpha-crystallin. Chemical crosslinking with bifunctional imido esters.

Reaction of calf alpha-crystallin, which consists of about 30 A and 10 B subunits, with various bisimido esters at pH 8.0-8.5 and room temperature leads to inter-subunit crosslinking. Little difference is observed in the efficiency of crosslinking by reagents of a homologous series with different lengths (from C6 to C12). At any stage of the reaction an exponential decrease is found in the amounts of crosslinked dimer, trimer, tetramer etc., with no preference for the formation of any particular oligomer, suggesting that all subunits on the surface of the spherical alpha-crystallin molecule are in quasi-equivalent positions. Molecular weight analysis by docdecylsulfate gel electrophoresis and reversible crosslinking show that both A and B subunits occur in an apparently constant ratio in the crosslinked oligomers, from which we infer that both types of subunits are randomly arranged on the surface of the alpha-crystallin molecule. Not all of the subunits can form inter-chain crosslinks, as a limiting value of about 60% of the subunits is found in oligomeric form. A simple explanation could be a core of subunits which is inaccessible to certain chemical reagents, although other alternatives are also discussed.

Biochemistry of human milk in early lactation.

With the analysis of more than 550 human milk samples we showed that triglycerides (accounting for 96-99% of the total lipids) increased from 2 to 3.5 g/100 ml mainly during the first week postpartum and remained constant thereafter. In contrast, both cholesterol and vitamin E concentrations decreased from 35 to 20 mg/100 ml and from 1.7 to 0.30 mg/100 ml, respectively. The phospholipids remained constant (40 mg/100 ml). Also the fatty acid composition of total lipids changed. Mid chain fatty acids (C10, C12, C14) increased, whereas the long chain polyunsaturated fatty acids decreased. The P/S-ratio of 0.32 remained constant throughout lactation. The mean protein content of human milk decreased from approximately 2 g/100 ml at day 2 to approximately 1 g/100 ml at day 36 of lactation. The content of each individual amino acid decreased likewise. However, when we expressed the amino acid values in relation to the protein value--that is as g amino acid per g protein--some decreased, some remained constant and some increased indicating a changing protein pattern. From these data we computer-calculated a sharp decrease in IgA content, a moderate decrease for lactoferrin and constant values for casein and a-Lactalbumin. Throughout the whole period of investigation, non protein nitrogen accounted for about 20% of total nitrogen. Although the absolute values decreased according to total nitrogen, the sum of free amino acids, as well as the amount of taurine, remained constant. Lactose increased from about 4 g/100 ml to 6 g/100 ml during the first days of lactation. The calcium content increased from a mean value of 25 mg/100 ml at day 1 to 32 mg/100 ml at day 5 and remained constant at 30 mg/100 ml up to day 36. Phosphorus content increased from 10 mg/100 ml at day 1 to 17 mg/100 ml at day 8 and then continuously decreased to 13 mg/100 ml at day 36. The protein-bound part of the calcium remained constant during the period investigation, the fat-associated part increased from 11% to 26%. Protein-bound phosphorus decreased from 45% in colostral milk to 29% in transitional and 23% in mature milk.

Comparative and age-dependent aspects of crystallin size and distribution in human, rabbit, bovine, rat, chicken, duck, frog and dogfish lenses.

Lens extracts obtained from eight vertebrate species were analyzed using high-performance gel permeation chromatography with differential refractive index and low-angle laser light scattering detection. The elution patterns enabled the direct determination of the proportions of the crystallin classes as well as the molecular weights of the numerous size fractions in a more rapid and accurate way than previously. Concerning the mammalian species, also age-dependent aspects could be assessed by comparing the data obtained from the extracts of differently aged lenses and/or lens parts.

High-performance gel permeation chromatography of bovine eye lens proteins in combination with low-angle laser light scattering. Superior resolution of the oligomeric beta-crystallins.

Calf lens extracts were subjected to high-performance gel permeation chromatography on TSK GEL G4000 SW and G3000 SW columns (fractionation range: 5 x 10(6)-10(4) daltons) and resolved into thirteen crystallin fractions: HM-, alpha-, six beta H-, two beta L-, beta S- and two gamma-crystallins. Molecular weights were determined using a low-angle laser light scattering detection system. The weight average and number average molecular weights for cortical alpha-crystallin, 860,000 and 740,000, respectively, reveal a polydispersity factor of 1.16 for this heterogeneous protein. The eight different beta-crystallin fractions could be found with practically all possible oligomeric structures from dimers to aggregates larger than dodecamers. Different structures are found for the predominant beta H-crystallin fractions, viz., hexamers and pentamers, in the extracts from cortex and nucleus. Additional identification of the fractions by sodium dodecyl sulphate gel electrophoresis and isoelectric focusing in the presence of urea also indicated that semi-preparative application of this high-performance technique is possible. The co-elution of putative cytoskeletal proteins with some beta-crystallins was remarkable; moreover, co-elution of FM-crystallin with beta S-crystallin is discussed. A 23,000-dalton fraction, mainly found in the cortical region, most likely corresponds to the 24,000-dalton gamma-crystallin preparation obtained from cattle lens cortices. It is questioned whether the similarities between this fraction and beta S-crystallin are merely coincidental.

Influence of long chain unsaturated fatty acids in formula feeds on lipid peroxidation and antioxidants in preterm infants.

The influence of long chain polyunsaturated fatty acids (LCP) in formula feeds on lipid peroxidation and antioxidants was studied in 35 healthy preterm infants (gestational age 30-35 wk) during the first 6 postnatal weeks. Infants received a preterm formula supplemented with n-3 LCP (LCP group, n = 13), or standard preterm formula (NO-LCP group, n = 15); 7 infants fed human milk served as a reference group. With LCP supplementation, erythrocyte C22:6n-3 levels were stable; without supplementation, the levels declined (difference p < 0.001). LCP supplementation did not decrease vitamin E or C levels, or increase lipid peroxidation products (thiobarbituric acid-reactive substances) in plasma. In erythrocytes, LCP supplementation did not markedly influence the reduced/oxidized glutathione ratio; however, the susceptibility to H2O2-induced oxidative stress was reduced. Our results suggest that healthy preterm infants are able to cope with any extra peroxidative stress produced by n-3 LCP supplementation. However, these findings might not be generally applicable to other formulas containing LCP supplements.

Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods.

BACKGROUND: An obvious difference between breast-fed and formula-fed newborn infants is the development of the intestinal flora, considered to be of importance for protection against harmful micro-organisms and for the maturation of the intestinal immune system. In this study, novel molecular identification methods were used to verify the data obtained by traditional culture methods and to validate the culture independent fluorescent in situ hybridization (FISH) technique. METHODS: From each of six breast-fed and six formula-fed newborn infants, six fecal samples were obtained during the first 20 days of life. The microbial compositions of the samples were analyzed by culturing on specific media and by FISH, by using specific 16S rRNA-targeted oligonucleotide probes. The colonies growing on the media were identified by random amplified polymorphic DNA pattern analysis and by polymerase chain reaction amplification and subsequent analysis of the 16S rRNA gene. RESULTS: Molecular identification of the colonies showed that the selective media are insufficiently selective and unsuitable for quantitative analyses. Qualitative information from the culturing results combined with the data obtained by the FISH technique revealed initial colonization in all infants of a complex (adult-like) flora. After this initial colonization, a selection of bacterial strains began in all infants, in which Bifidobacterium strains played an important role. In all breast-fed infants, bifidobacteria become dominant, whereas in most formula-fed infants similar amounts of Bacteroides and bifidobacteria (approximately 40%) were found. The minor components of the fecal samples from breast-fed infants were mainly lactobacilli and streptococci; samples from formula-fed infants often contained staphylococci, Escherichia coli, and clostridia. CONCLUSIONS: This study confirms the differences in development of intestinal flora between breast-fed and formula-fed infants. The results obtained from the FISH technique were consistent. Although the repertoire of probes for this study was not yet complete, the FISH technique will probably become the method of reference for future studies designed to develop breast-fed-like intestinal flora in formula-fed infants.