A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

Amino-acid enzyme activities in liver and kidney of developing rats

The amino-acid enzymes (aspartate-, alanine- and tyrosine transaminases, serine dehydratase, glutamate dehydrogenase, glutamine synthetase, adenylate deaminase and arginase) activities in the liver and kidney of developing rats (days 19 and 21 after conception and 1, 5, 10, 20 and 30 after birth) compared with adults were determined in crude homogenates. Most enzymes attained the adult levels early after birth or at weaning, showing a marked trend towards amino-acid nitrogen conservation during late foetal and specially during the neonatal period, increasing their activity during lactation. It is postulated that these changes are closely related to availability of low grade protein in diet as well as to maturation of amino-acid homeostasis maintenance for growth.     

Metabolic utilization of muscular L-proline in 24-hr starved rats.

1. The aim of this paper was to study the in vivo skeletal muscle L-proline related to its destination to other key tissues such as liver and intestine as well as to give some insight into the role of blood cells in proline handling. 2. L-U-[14C]Proline was injected intramuscularly and following by sampling of blood, liver, intestine and contralateral muscle at 20 and 30 min after injection. 3. The distribution of radioactivity between blood cells and plasma and in total and individual amino acids, protein and glycogen fractions was determined in the above tissues. 4. The pattern of well fed rats was compared with those submitted to 24-hr complete starvation. 5. During starvation a minor degree of proline oxidation occurs. 6. The main destruction of proline in the liver seem to be the synthesis of proteins. 7. The radioactivity recovered in the blood proline fraction of starved rats is twice that of the fed rats and that it could be attributed mainly to plasma protein. 8. We have obtained in vivo evidence for the role of erythrocyte in the interorgan proline transport.     

A contribution to the phenotype distribution of phosphoglucomutase in Czechoslovakia (the district of České Budějovice)

Summary A population sample of 416 unrelated donors from eské Budjovice (southorn Bohemia) was investigated for the phenotypes of phosphoglucomutase (PGM). The calculated frequencies of the allelles PGM ₁ ¹ and PGM ₁ ² , 0.770 and 0.230, respectively, correspond to the expected frequencies of the phenotypes PGM 1=0.593, PGM 2-1=0.354, and PGM 2=0.0529. No rare phonotype was detected.     

Pharmacological and nutritional agents promoting browning of white adipose tissue

The role of brown adipose tissue in the regulation of energy balance and maintenance of body weight is well known in rodents. Recently, interest in this tissue has re-emerged due to the realization of active brown-like adipose tissue in adult humans and inducible brown-like adipocytes in white adipose tissue depots in response to appropriate stimuli (“browning process”). Brown-like adipocytes that appear in white fat depots have been called “brite” (from brown-in-white) or “beige” adipocytes and have characteristics similar to brown adipocytes, in particular the capacity for uncoupled respiration. There is controversy as to the origin of these brite/beige adipocytes, but regardless of this, induction of the browning of white fat represents an attractive potential strategy for the management and treatment of obesity and related complications. Here, the different physiological, pharmacological and dietary determinants that have been linked to white-to-brown fat remodeling and the molecular mechanisms involved are reviewed in detail. In the light of available data, interesting therapeutic perspectives can be expected from the use of specific drugs or food compounds able to induce a program of brown fat differentiation including uncoupling protein 1 expression and enhancing oxidative metabolism in white adipose cells. However, additional research is needed, mainly focused on the physiological relevance of browning and its dietary control, where the use of ferrets and other non-rodent animal models with a more similar adipose tissue organization and metabolism to humans could be of much help. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N ^ε-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.     

Mitochondrial implications in human pregnancies with intrauterine growth restriction and associated cardiac remodelling

Intrauterine growth restriction (IUGR) is an obstetric complication characterised by placental insufficiency and secondary cardiovascular remodelling that can lead to cardiomyopathy in adulthood. Despite its aetiology and potential therapeutics are poorly understood, bioenergetic deficits have been demonstrated in adverse foetal and cardiac development. We aimed to evaluate the role of mitochondria in human pregnancies with IUGR. In a single‐site, cross‐sectional and observational study, we included placenta and maternal peripheral and neonatal cord blood mononuclear cells (PBMC and CBMC) from 14 IUGR and 22 control pregnancies. The following mitochondrial measurements were assessed: enzymatic activities of mitochondrial respiratory chain (MRC) complexes I, II, IV, I + III and II + III, oxygen consumption (cell and complex I‐stimulated respiration), mitochondrial content (citrate synthase [CS] activity and mitochondrial DNA copy number), total ATP levels and lipid peroxidation. Sirtuin3 expression was evaluated as a potential regulator of bioenergetic imbalance. Intrauterine growth restriction placental tissue showed a significant decrease of MRC CI enzymatic activity (P < 0.05) and CI‐stimulated oxygen consumption (P < 0.05) accompanied by a significant increase of Sirtuin3/β‐actin protein levels (P < 0.05). Maternal PBMC and neonatal CBMC from IUGR patients presented a not significant decrease in oxygen consumption (cell and CI‐stimulated respiration) and MRC enzymatic activities (CII and CIV). Moreover, CS activity was significantly reduced in IUGR new‐borns (P < 0.05). Total ATP levels and lipid peroxidation were preserved in all the studied tissues. Altered mitochondrial function of IUGR is especially present at placental and neonatal level, conveying potential targets to modulate obstetric outcome through dietary interventions aimed to regulate Sirtuin3 function.