Aldehyde dismutase activity of human liver alcohol dehydrogenase.

Human alcohol dehydrogenases of class I and class II but not class III catalyse NAD+-dependent aldehyde oxidation in addition to the NADH-dependent aldehyde reduction. The two reactions are coupled, i.e. the enzymes display dismutase activity. Dismutase activity of recombinantly expressed human class I isozymes beta1beta1 and gamma2gamma2, class II and class III alcohol dehydrogenases was assayed with butanal as substrate by gas chromatographic-mass spectrometric quantitations of butanol and butyric acid. The class I gamma2gamma2 isozyme showed a pronounced dismutase activity with a high kcat, 1300 min(-1), and a moderate Km, 1.2 mM. The class I beta1beta1 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower kcat values, 60-75 min(-1). 4-Methylpyrazole, a potent class I ADH inhibitor, inhibited the class I dismutation completely, but cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, did not affect the dismutation. The dismutase reaction might be important for metabolism of aldehydes during inhibition or lack of mitochondrial aldehyde dehydrogenase activity.

Amino acid profiles by HPLC after filter paper sampling: 'appropriate technology' for monitoring of nutritional status.

This paper describes a rapid analysis of free amino acid levels in capillary blood samples using a modified HPLC system. Capillary whole blood (25 microliters) is dried on a filter paper, extracted and the equivalent of 0.25 microliter of the initial blood sample is used for each amino acid analysis. Nineteen free amino acid levels are determined with a reproducibility of better than +/- 10% for the entire procedure of sampling, preparation and analysis, with the exception of ornithine (+/- 19%) and lysine (+/- 12%). Cystine and proline cannot easily be determined by this method. Alanine, tyrosine, methionine, valine, phenylalanine, isoleucine and leucine concentrations on the filter paper are unaltered after 1, 2 and 21 wk. Storage at room temperature should not be for longer than 2 wk, but storage at +4 degrees C, -18 degrees C and -70 degrees C is acceptable for 21 wk. This new micromethod seems to be a practical and reliable tool. Because of its simplicity and, above all, the need for a minimal amount of capillary blood, it is a valid means for the routine monitoring of amino acid profiles in sick preterm infants on different protein regimens. The sampling and storage methods are also examples of 'appropriate technology' for field studies of nutritional adequacy in population samples derived from infants. This is because centrifugation is not necessary and the fact that the relevant amino acids on the dried filter paper samples display high stability.

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

Blood levels of critical amino acids in very low birthweight infants on a high human milk protein intake.

A method for a semi-industrial production of human milk subfractions (human milk protein and human milk fat isolates) is described. Four very low birthweight (VLBW) newborn were given a human milk protein isolate added to the mother's own fresh expressed milk in addition to sodium chloride up to 20 mEkv/liter. Growth followed the intrauterine growth curve. Urea levels did not increase in spite of providing a double-normal protein intake. There was no metabolic acidosis and the blood levels of free amino acids determined with a micro-method did not exceed those seen after a normal meal. The concentrated human milk protein product showed a considerable specific sIgA activity against E. coli 0-antigen. It seems possible to use similar "lacto-engineering"-techniques in order to satisfy the increased protein requirements of the VLBW infant, while providing the caloric requirements, without causing any visible disturbance of blood-homeostasis of urea, amino acids or base excess. The method could provide knowledge about the "human milk protein requirements" and a controlled study has been started.

Absorption of biological amines of bacterial origin in normal and sick infants.

This investigation aims at monitoring the formation of diamines in the gastrointestinal tract of human infants, and thereby also the bacterial colonization of the intestine, by studying the urinary excretion of the heterocyclic amine piperidine during development and in different malabsorptive states. A gas chromatographic assay of the dinitrophenyl derivative of piperidine and a mass spectrometric method of identification were worked out and applied. The piperidine excretion was expressed in units of urinary creatinine concentration. Adult women show a greater variation than men, both inter- and intra-individually. The piperidine excretion is very low and mostly undetectable in the first week of life. There is an increase in weaning, with a significant difference between breast-fed and formula-fed infants at 4--6 months. There is a significant difference between infants suffering from untreated coeliac disease and infants without malabsorption. The findings indicate that piperidine excretion is a sensitive biochemical index of changes in the gastrointestinal flora. The high excretion in coeliac disease suggests that piperidine, which is known to have nicotine-like synaptic activity in the CNS, is one of the hitherto unidentified 'auto-intoxicating' substances arising from the bacterial decomposition of protein suggested by Metchnikoff in 1903.

Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin.

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gammagamma ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min-1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5-HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 microm; kcat = 40 min-1). In a system where NAD+ is regenerated, the oxidation of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid proceeds with concentration levels of the intermediary 5-HIAL expected for a two-step oxidation. Butanal and 5-HIAL oxidation is also observed for class-I ADH in the presence of NADH. The class-II enzyme is less efficient in aldehyde oxidation, and the ethanol-oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5-hydroxyindole-3-acetic acid (Ki = 2 mm). Reduction of 5-HIAL is efficiently catalysed by class-I gammagamma ADH (kcat = 400 min-1; Km = 33 microm) in the presence of NADH. This indicates that the increased 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class-I ADH.

Plasma valine and urinary C-peptide in breast-fed and artificially fed infants up to 6 months of age.

Plasma branched-chain amino acids and urinary C-peptide-creatinine excretion was determined at 3, 4 1/2 and 6 months of age in a group of 50 infants who were either breast-fed or artificially fed and selected at random. The average concentrations of valine in plasma and C-peptide in urine as well as the ratio between C-peptide and creatinine in urine were 2-3 times higher (p less than 0.01) in artificially fed as compared to breast-fed infants at all the ages studied. Plasma valine values correlated significantly with the urinary C-peptide/creatinine ratio (r = 0.76, p less than 0.01), which suggests that the enhanced insulin response induced by the artificial formula is related to its protein content.

The protein tolerance of very low birth weight infants fed human milk protein enriched mother's milk.

Pooled breast milk was ultra-filtrated and freeze-dried to give a product with a protein content of 60 g/100 g powder. More than half of the secretory IgA activity against E. coli O antigen was preserved. This concentrated protein was added to the mother's fresh milk providing a protein supply of 3.0-3.5 g/kg/24 hours in four VLBW infants, at a calorie supply of 110 kcal/kg/24 hours. Growth followed the intrauterine rate. Free amino acid levels, acid-base balance and urea concentrations of peripheral whole blood indicated tolerance of the increased supply of human milk protein.