Low Protein Formula: Consequences of Quantitative Effects of Pre-analytical Factors on Amino Acid Concentrations in Plasma of Healthy Infants.

OBJECTIVE: Quantifying pre-analytical effects of postprandial sampling delay and daily protein intake on plasma amino acid concentrations in healthy infants fed formula with low protein content (1.8-1.9 g/100 kcal). Intake of formula with higher protein content bears a risk for later obesity (Kirchberg, J Clin Endocrinol Metab 100(1):149-158, 2015). Formulas containing less than 1.8 g protein might be adequate but not safe (Fomon, J Pediatr Gastroenterol Nutr 28:495-501, 1999). With on-demand feeding reproducible controls of indispensible amino acid concentration cannot be made at trough level. METHODS: Data of 102 healthy infants aged 1 month and 79 aged 4 months fed formula with low protein content were obtained from a previous study (Haschke-Becher, J Inherit Metab Dis 39(1):25-37, 2016). They were analysed by multiple regression. Independent variables were the postprandial sampling delay from 2.25 to 4.5 h and the daily protein intake. Dependant variables were the amino acid concentrations. The combined effect was calculated with the natural logarithm of the amino acid concentration. RESULTS: Most amino acids fitted a significant exponential decrease due to the sampling delay, except of aspartate, citrulline, glutamine, glutamate, histidine, tryptophan and tyrosine at 1 month; and at 4 months except of citrulline, glutamine, glutamate, glycine and ornithine. Significant effects of protein intake were found for lysine and serine at 1 month and for glutamate at 4 months of age. Lowest limits of significant amino acid concentrations were calculated by extrapolation of sampling delay to 5 h and using the 10th percentile after back-transformation to µmol/L. A procedure to avoid the pitfall of overestimating amino acid concentration is presented.

Reference values of amino acids and of common clinical chemistry in plasma of healthy infants aged 1 and 4 months.

OBJECTIVE: To compare plasma levels of amino acids and clinical chemistry parameters in healthy infants at 1 and 4 months of age and to establish corresponding reference limits. METHODS: Data of three multicenter studies assessing the safety of new infant formulas were used. During these studies infants of both age-groups were either breast-fed or received formulas of low or high protein content. All samples were analyzed centrally in the same accredited laboratory. RESULTS: Plasma was collected from 521 infants in total, 157 boys and 135 girls aged 1 month and 121 boys and 108 girls aged 4 months. At the age of 1 month, 62 infants had received exclusively breast milk, 198 exclusively formula, and 27 both; in the 4-months age group corresponding numbers were 49, 158 and 18, respectively; for 9 infants, diet was unknown. Concentrations of most amino acids and clinical chemistry parameters differed significantly between both ages. Regardless of age, most plasma amino acid levels were comparable or lower in breast-fed than in formula-fed infants whereas at 1 month of age most clinical chemistry parameters were higher. While in breast-fed infants the plasma urea concentration decreased over 4 months of age, it increased in formula-fed infants. There were significant differences between infants fed a low and high protein formula. At both ages, high protein formulas resulted in significantly higher threonine, 2-aminobutyrate, and urea concentrations. CONCLUSIONS: For clinical use, age- and diet specific reference limits in infants are warranted.

Efficient subtractive cloning of genes activated by lipopolysaccharide and interferon γ in primary-cultured cortical cells of newborn mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders.

Urinary D-lactate excretion in infants receiving Lactobacillus johnsonii with formula.

BACKGROUND/AIMS: Supplementation with certain probiotics can improve gut microbial flora and immune function but should not have adverse effects. This study aimed to assess the risk of D-lactate accumulation and subsequent metabolic acidosis in infants fed on formula containing Lactobacillus johnsonii (La1). METHODS: In the framework of a double-blind, randomized controlled trial enrolling 71 infants aged 4-5 months, morning urine samples were collected before and 4 weeks after being fed formulas with or without La1 (1 x 10(8)/g powder) or being breastfed. Urinary D- and L-lactate concentrations were assayed by enzymatic, fluorimetric methods and excretion was normalized per mol creatinine. RESULTS: At baseline, no significant differences in urinary D-/L-lactate excretion among the formula-fed and breastfed groups were found. After 4 weeks, D-lactate excretion did not differ between the two formula groups, but was higher in both formula groups than in breastfed infants. In all infants receiving La1, urinary D-lactate concentrations remained within the concentration ranges of age-matched healthy infants which had been determined in an earlier study using the same analytical method. Urinary L-lactate also did not vary over time or among groups. CONCLUSIONS: Supplementation of La1 to formula did not affect urinary lactate excretion and there is no evidence of an increased risk of lactic acidosis.

Interpretation of plasma amino acids in the follow-up of patients: the impact of compartmentation.

Results of plasma or urinary amino acids are used for suspicion, confirmation or exclusion of diagnosis, monitoring of treatment, prevention and prognosis in inborn errors of amino acid metabolism. The concentrations in plasma or whole blood do not necessarily reflect the relevant metabolite concentrations in organs such as the brain or in cell compartments; this is especially the case in disorders that are not solely expressed in liver and/or in those which also affect nonessential amino acids. Basic biochemical knowledge has added much to the understanding of zonation and compartmentation of expressed proteins and metabolites in organs, cells and cell organelles. In this paper, selected old and new biochemical findings in PKU, urea cycle disorders and nonketotic hyperglycinaemia are reviewed; the aim is to show that integrating the knowledge gained in the last decades on enzymes and transporters related to amino acid metabolism allows a more extensive interpretation of biochemical results obtained for diagnosis and follow-up of patients and may help to pose new questions and to avoid pitfalls. The analysis and interpretation of amino acid measurements in physiological fluids should not be restricted to a few amino acids but should encompass the whole quantitative profile and include other pathophysiological markers. This is important if the patient appears not to respond as expected to treatment and is needed when investigating new therapies. We suggest that amino acid imbalance in the relevant compartments caused by over-zealous or protocol-driven treatment that is not adjusted to the individual patient's needs may prolong catabolism and must be corrected.

Expression and function of AGAT, GAMT and CT1 in the mammalian brain.

In mammals, creatine is taken up from the diet and can be synthesized endogenously by a two-step mechanism involving the enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Creatine (Cr) is taken up by cells through a specific transporter, CT1. While the major part of endogenous synthesis of Cr is thought to occur in kidney, pancreas and liver, the brain widely expresses AGAT, GAMT and CT1, both during development and in adulthood. The adult central nervous system (CNS) has a limited capacity to take up Cr from periphery, and seems to rely more on its endogenous Cr synthesis. In contrast, the embryonic CNS might be more dependent on Cr supply from periphery than on endogenous synthesis. This review will focus on the expression and function of AGAT, GAMT and CT1 in the mammalian CNS, both during development and in adulthood. Emphasis will also be placed on their specific roles in the different cell types of the brain, to analyze which brain cells are responsible for the CNS capacity of (i) endogenous Cr synthesis and (ii) Cr uptake from the periphery, and which brain cells are the main Cr consumers. The potential role of CT1 as guanidinoacetate transporter between "AGAT-only" and "GAMT-only" expressing cells will also be explored.

Creatine synthesis and transport during rat embryogenesis: spatiotemporal expression of AGAT, GAMT and CT1.

BACKGROUND: Creatine (Cr) is synthesized by a two-step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), and is taken up by cells through a specific Cr transporter, CT1. Recently, genetic defects of this pathway have been described, that lead to Cr deficiency, neurological symptoms in early infancy and severe neurodevelopmental delay. To investigate the involvement of Cr synthesis and uptake pathways during embryonic development, we determined the spatiotemporal expression of AGAT, GAMT and CT1 during the rat embryogenesis, at the mRNA and protein level. RESULTS: We show that AGAT and GAMT are expressed in hepatic primordium as soon as 12.5 days, then progressively acquire their adult pattern of expression, with high levels of AGAT in kidney and pancreas, and high levels of GAMT in liver and pancreas. AGAT and CT1 are prominent in CNS, skeletal muscles and intestine, where they appear earlier than GAMT. High levels of CT1 are found in epithelia. CONCLUSION: Our results suggest that de novo synthesis of Cr by AGAT and GAMT, as well as cellular Cr uptake by CT1, are essential during embryonic development. This work provides new clues on how creatine can be provided to developing tissues, and suggests that Cr deficiencies might induce irreversible damages already in utero, particularly on the nervous system.

Ammonia toxicity to the brain and creatine.

Symptoms of hyperammonemia are age-dependent and some are reversible. Multiple mechanisms are involved. Hyperammonemia increases the uptake of tryptophan into the brain by activation of the L-system carrier while brain glutamine plays a still undefined role. The uptake of tryptophan by the brain is enhanced when the plasma levels of branched-chain amino acids competing with the other large neutral amino acids are low. Hyperammonemia increases the utilization of branched-chain amino acids in muscle when ketoglutarate is low, and this is further enhanced by glutamine depletion (as a result of therapy with ammonia scavengers like phenylbutyrate). Anorexia, most likely a serotoninergic symptom, might further aggravate the deficiency of indispensable amino acids (e.g., branched-chain and arginine). The role of increased glutamine production in astrocytes and the excitotoxic and metabotropic effects of increased extracellular glutamate have been extensively investigated and found to differ between models of acute and chronic hyperammonemia. Using an in vitro model of cultured embryonic rat brain cell aggregates, we studied the role of creatine in ammonia toxicity. Cultures exposed to ammonia before maturation showed impaired cholinergic axonal growth accompanied by a decrease of creatine and phosphocreatine, a finding not observed in mature cultures. By using different antibodies, we have shown that the phosphorylated form of the intermediate neurofilament protein is affected. Adding creatine to the culture medium partially prevents impairment of axonal growth and the presence of glia in the culture is a precondition for this protective effect. Adequate arginine substitution is essential in the treatment of urea cycle defects as creatine is inefficiently transported into the brain.

Long-term outcome of patients with urea cycle disorders and the question of neonatal screening.

UNLABELLED: With regard to the principles established for neonatal population screening, the question arises whether for patients with urea cycle disorders there is an accepted treatment which really affects the disease course and prognosis as compared to the natural history of these diseases. A retrospective study of 88 patients was performed. Based on questionnaires, the survival rate and neurodevelopmental outcome of patients treated with protein restriction alone was compared to the new therapy introduced in the 1980s with added citrulline/arginine, essential amino acids for improving the amino acid composition of the restricted natural protein and benzoate. Survival of patients with neonatal presentation was improved by the extensive therapy but this mostly at the cost of an increasing number of retarded patients. Long-term outcome did not differ significantly between the two treatments. Of all patients, 56% were symptomatic within 4 days of age and 67% within the 1st week. Thus a prevention of irreversible damage by neonatal screening on blood obtained at 3-4 days of life is questionable. Whether the benefit of obtaining a rapid diagnosis, e.g. for allowing proper counselling and prospective treatment, is acceptable for the parents of prospective patients remains open. The organisation of a dense network of specialised metabolic centres with sufficient staff and resources is a prior condition for any screening programme in order to ensure the rapid diagnosis, follow-up of treatment and counselling of a cumulative number of affected chronic patients needing this support. A commitment on a long-term basis by the institutions is needed in view of the health budget restrictions. CONCLUSION: in the short term, the goal is to detect hyperammonaemic patients as early as possible with special emphasis on sick neonates. In practice, quantitative plasma ammonia determination without delay is recommended in any newborn for whom a sepsis work-up is considered and in children who refuse feeding or vomit and show alterations of consciousness and/or neurological symptoms.

Serum paracetamol concentration: an alternative to X-rays to determine feeding tube location in the critically ill.

BACKGROUND: Placement of enteral feeding tubes is an important clinical issue. Previous studies suggest that paracetamol absorption is very fast after jejunal administration. The aim was to determine whether paracetamol serum concentration measured by immunoassay can determine the tip position of the feeding tube. METHODS: Thirty-three critically ill patients requiring enteral nutrition with either gastric or post-pyloric feeding tubes were enrolled prospectively in the surgical intensive care unit of a university hospital. Paracetamol was administered in the feeding tube (15 mg/kg) after a baseline blood sample (T0). Thereafter, 8 blood samples were taken between 2.5 and 240 minutes. Paracetamol was analyzed using an automated homogenous immunoassay. RESULTS: The patients did not differ with respect to age or severity of disease. Peak paracetamol concentrations were significantly higher after post-pyloric administration with 42.6 +/- 13.5 versus 20.5 +/- 7.5 mg/L (p < .0001). Time-to-peak paracetamol concentration was significantly shorter with post-pyloric tubes (median, 5 versus 60 minutes; p < .0001). The receiver operating characteristic (ROC) curves showed the highest sensitivity and specificity at 5 minutes with 94.1% and 100%, respectively, for discriminating between gastric and post-pyloric location. CONCLUSIONS: Because of paracetamol's rapid absorption after jejunal administration, the test seems to be a safe and inexpensive alternative to X-ray control for assessment of the enteral feeding tube location. Its value in clinical practice remains to be established.

Outcome and survival of 88 patients with urea cycle disorders: a retrospective evaluation.

UNLABELLED: This paper presents data obtained by questionnaires sent to local hospitals and metabolic centres in Germany, Austria, Italy and Switzerland concerning the survival and outcome of patients with urea cycle disorders treated between 1975 and 1986. A total of 130 questionnaires were sent out of which 88 questionnaires of patients were returned. This study compares results of conservative long-term management using protein restriction versus protein restriction combined with more extensive treatment (arginine/citrulline, essential amino acid supplements and sodium benzoate as alternate pathway therapy). While survival was improved in neonates receiving extensive therapy, there was an increased risk of mental retardation on long-term follow-up in this group. The time at which the first symptoms of hyperammonaemia appeared, the age at diagnosis, and the time of delay in diagnosis after the first symptom were not helpful in predicting outcome; however, when plasma ammonia concentrations exceeded 300 micromol/l initially or 480 micromol/l at its peak, none of the patients had a normal cognitive outcome. CONCLUSION: there is a need to establish improved treatment approaches and a network of "rare disease" centres to assure the rapid diagnosis and effective treatment and follow-up of affected children. This should precede any consideration of neonatal screening for urea cycle disorders.

Organic acids in the second morning urine in a healthy Swiss paediatric population.

Organic acid analysis is used for the early detection/ exclusion and for the follow-up of inherited disorders of amino acid and organic acid metabolism. Urinary organic acid concentrations in 417 healthy Caucasian children (1 day to 17 years of age) were determined after liquid solid extraction, as their trimethylsilyl derivatives, by gas chromatography and mass spectrometry. Concentrations of most of the organic acids adjusted for creatinine tend to decrease with age. No differences were found between gender except for the Krebs cycle intermediates in the older age groups. In neonates, the immaturity of the neonatal kidney led to a much larger variation of organic acid levels when related to creatinine. The low number of subjects (n = 36-52) per age class resulted in large 95% confidence intervals of the percentiles used for decision. This must be taken into account when using the data for exclusion or diagnosis of disorders.

Infantile citrullinemia caused by citrin deficiency with increased dibasic amino acids.

In an infant who suffered from prolonged icterus and hepatocellular dysfunction we detected an increase of citrulline and dibasic amino acids in plasma and urine. The amino acid levels along with all the abnormal liver tests normalized upon replacing breast-milk by formula feeding; there was no relapse after human milk was tentatively reintroduced. A novel mutation, a approximately 9.5-kb genomic duplication, was identified in the citrin gene (SLC25A13) resulting in the insertion of exon 15. No mutation was detected in the CAT2A specific exon of the SLC7A2 gene which encodes for the liver transporter of cationic amino acids. This is the first report of infantile citrin deficiency in non-Asian patients.

Ammonium-induced impairment of axonal growth is prevented through glial creatine.

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

N-acetylglutamate synthase deficiency and the treatment of hyperammonemic encephalopathy.

Carbamylphosphate synthase is the first enzymatic reaction of the urea cycle. Its activator, N-acetylglutamate, is synthesized from acetyl-CoA and glutamate in a reaction catalyzed by N-acetylglutamate synthase (NAGS). We have identified the putative human NAGS gene and report the first mutation in this gene in a family with carbamylglutamate responsive hyperammonemia and normal activity of the urea cycle enzymes. Mutation analysis has a higher diagnostic specificity than the enzymatic assay in NAGS deficiency. A therapeutic trial with carbamylglutamate is recommended whenever hyperammonemia without an organic aciduria, increased orotate excretion, or diagnostic amino acidemia/uria is detected.

Whey predominant, whey modified infant formula with protein/energy ratio of 1.8 g/100 kcal: adequate and safe for term infants from birth to four months.

BACKGROUND: Protein quality of breast milk is superior to that of formula proteins. To ensure that the protein intake is sufficient, starter formulas with conventional protein composition provide a protein/energy ratio of 2.2-2.5 g per 100 kcal to infants, which is much higher than that supplied with breast milk. Several studies have shown that formula-fed infants have higher plasma or serum urea concentrations than breast-fed infants do. We tested if feeding formulas with improved protein quality and a protein content corresponding to the minimum level that is consistent with international recommendations (1.8 g/100 kcal) allows patients to achieve normal growth and plasma urea concentrations. METHODS: Healthy term infants were enrolled into the study and were either breast-fed or randomly assigned to three formula-fed groups. Formula-fed infants received either a standard formula with a protein/energy ratio of 2.2 g/100 kcal, whereas the two other groups received formulas with a protein/energy ratio of 1.8g/100 kcal differing mainly by their source of protein. Subjects received breast milk or these formulas ad libitum as the sole source of energy from birth to four months of age in a controlled blind design (except for the breast-fed group). Anthropometric measurements (body weight and length) were obtained at birth, at 30, 60, 90, and 120 days. Energy and protein intakes were calculated from three-day dietary records. Blood was collected for biochemical measurements at 30, 60, and 120 days. RESULTS: No differences were found between the four feeding groups for weight- and length-gains or for body mass indices (BMI). No differences in energy intakes between the formula-fed groups could be found, whereas protein intakes were less in infants fed the 1.8 g/100 kcal formulas. Plasma urea levels of the infants fed the 1.8 g/100 kcal formulas were closer to those found in the breast-fed infants. CONCLUSION: Improvement of the amino acid profile permits a whey predominant starter formula with 1.8 g protein per 100 kcal to meet the needs of normal term infants during the first four months of life.

Mechanisms of hyperammonemia.

Hyperammonemia is mainly found in hepatic encephalopathy and in genetic defects of the urea cycle or other pathways of the intermediary metabolism. Clinically a difference has to be made between chronic moderate hyperammonemia and acutely increased concentrations. Pathogenetic mechanisms of ammonia toxicity to the brain are partly unraveled. In some animal models confounding variables, such as the reduced intake of food and amino acid imbalance due to liver insufficiency, do not allow to establish unequivocal causal relationships between the ammonia concentration and measured effects. In chronic moderate hyperammonemia an increased flux through the serotonin pathway is a key factor. It is caused by an increased transport of large neutral amino acids (including tryptophan) through the blood-brain barrier, accentuated by the imbalance of plasma amino acids in hepatic insufficiency. It is stimulated by D- or L-glutamine. Evidence is presented showing that a functioning gamma-glutamyl cycle (glutathione formation) is a prerequisite. In acute hyperammonemia involvement of NMDA receptors, glutamate, NO and cGMP plays an additional role. In hyperammonemic crises the increased cerebral blood flow leads to brain edema; factors discussed here are increased osmolytes in astrocytes and serotoninergic activity. Recent data indicate that axonal development is affected by ammonia and can be normalized in vitro by creatine supplementation in developing mixed brain cell aggregate cultures, thus reviving the old hypothesis of the impact of hyperammonemia on energy metabolism in the developing brain that could cause mental retardation.

Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions.

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.