Effect of experimental hyperphenylalaninemia induced by dietary phenylalanine plus α-methylphenylalanine administration on amino acid concentration in neonatal chick brain,plasma, and liver

Supplementation of 5% phenylalanine plus 0.4% -methylphenylalanine to the standard diet or 1% phenylalanine plus 0.08% -methylphenylalanine to the drinking water produced phenylketonuria-like conditions in 5-day-old chicks. An increase of 10 to 15-fold in the phenylalanine content was observed in plasma or brain of animals after 9 days of both types of treatment. A smaller but significant increase was also observed in liver. However, practically no changes were found in the levels of tyrosine in the same conditions. Thus, the high values of plasma and brain phenylalanine/tyrosine ratio obtained by these treatments were mainly due to an increase in the phenylalanine levels, without increasing those of tyrosine. Chronic hyperphenylalaninemia induced a nonsignificant decrease in the most of amino acid contents in brain, especially after 9 days of treatment, although the levels of glycine and serine were significantly increased. A similar decrease was found in the plasma and liver concentration of various amino acids, although the variations observed in the liver were smaller than those found in plasma and brain.     

Nucleoside incorporation as a function of hormone levels during the early phases of estrogen-induced genesis of medullary bone in the Japanese quail,Coturnix coturnix

Summary The sequence of changes in RNA synthesis during the early phases of genesis of medullary bone induced in male Japanese quail by estrogen treatment was studied by ³H-uridine uptake. Analyses of plasma estrogen and testosterone were done by radioimmunoassay at 12, 24, 38 and 61 h. A dose of 5 mg kg^-1 estradiol-17 was found to stimulate the same ³H-uridine uptake 15 h after hormone treatment as a dose of 20 mg kg^-1 of estradiol valerate. The uptake of ³H-uridine rose as the dose of estradiol-17 increased. Plasma estrogen levels, which were highest 12 h after injection, declined sharply during the next 12 h, returning to control levels between 38 and 61 h. Testosterone levels declined after estrogen administration and remained below control values at all time points. Following estrogen administration, ³H-uridine uptake declined from control values for the first 8 h. Twelve hours after hormone administration control levels were again reached, with maximum ³H-uridine uptake occurring 16 h after hormone treatment. The 16-h maximum was followed by a steady decline to below control levels at 20, 24 and 28 h, the time at which the experiment was discontinued. Maximum ³H-uridine up-take following estrogen stimulation is similar to that observed for the stimulated immature rat uterus.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

An extended developmental study of γ-glutamyltranspeptidase in rat liver plasma membranes: identification of specific patterns of changes in activity in the adult as well as the neonatal state

Summary Homogenates and plasma membranes were isolated from the livers of male Fischer 344 rats ranging in age from 19 hr to 92 days postnatal. These plasma membranes exhibited comparable levels of purity: protein yields were 2–2.5%; relative specific activities of 5-nucleotidase and ouabain-sensitive Na⁺/K⁺-ATPase were from 8–11 and from 12–19, respectively. 5-nucleotidase and ouabain-sensitive Na⁺-K⁺-ATPase displayed distinct and different developmental patterns. The activity of -glutamyltranspeptidase was found to be at exceptionally high levels in isolated plasma membranes immediately after birth and to decline precipitously thereafter achieving and maintaining low levels from days 3–21 postnatal. Liver plasma membrane -glutamyltranspeptidase activity was observed to increase 9.2 fold from this low point, first rising on day 21, peaking on day 40 and returning to low levels by day 56. From day 56 day to 92 postnatal, -glutamyltranspeptidase activity was expressed at a uniformly low level but a level 2 fold higher than that preceeding the rise at day 40. The hormone determinants of these developmental changes in -glutamyltranspeptidase activity are discussed.     

Intracellular concentrations of phenylalanine,tyrosine and α-aminobutyric acid in 13 homozybotes and 19 heterozygotes for phenylketonuria (PKU) compared with 26 normals

Summary Intracellular concentrations of phenylalanine, tyrosine, -aminobutyric acid, and seven other aminoacids (glycine, alanine, valine, cystine, methionine, isoleucine, leucine) were measured in lymphocytes of 13 homozygotes and 19 heterozygotes for phenylketonuria and in lymphocytes of 26 normals. Intracellular concentrations for phenylalanine, tyrosine, and -aminobutyric acid were significantly higher in homo- and heterozygotes than in normals (P<0.001; P<0.01). For the other seven aminoacids there were no or only questionable differences. Between homo-and heterozygotes there was no difference in any of the aminoacids. The intracellular phenylalanine: tyrosine ratio was essentially the same in all three groups of individuals. There was no correlation between intracellular phenylalanine above or below 10nmol/10⁶ cells and IQ in heterozygotes. The same is true for phenylalanine: tyrosine ratio greater or smaller than 1. In homozygotes there was no correlation between intracellular phenylalanine and age—to which DQ/IQ is correlated. There was no significant difference in intracellular phenylalanine between homozygotes with blood levels above and below 908 mol/l (15 mg/100 ml) at the time of blood sampling and no correlation between intra- and extracellular phenylalanine concentrations.Among the 26 normals there were only two with intracellular phenylalanine above 10 nmol/10⁶ cells, both showing phenylalanine loading test curves suggestive of heterozygosity.The results are discussed and important functions of the cell wall are proposed. The formation of an abnormal unknown intracellular metabolite being the real noxious agent could explain the incomparably different degrees of brain dysfunction in individuals with equal though elevated intracellular phenylalanine concentrations, i.e., homozygotes and heterozygotes for PKU.     

Time-course of changes in plasma levels of trace elements after thrombolysis during the acute phase of myocardial infarction in humans

It has been suggested that the injury induced by reperfusion of the ischemic myocardium could result, in part, from the cytotoxic effects of oxygen free radicals. Since various trace elements are involved in several of the reactions leading to free radical production, we have measured plasma levels of copper, zinc, selenium, and iron:

1. In 18 patients (mean age 60 yr old) subjected to thrombolytic therapy within 6 h after the onset of a myocardial infarction (G1);
2. In 16 patients with coronary artery disease, but without a history of a previous myocardial infarction (MI) (mean age 50 yr old, G2); and
3. In 50 healthy volunteers divided into two subgroups according to age (mean age 33 yr old, G3 and 55 yr old, G4).

Plasma myosin levels were used to estimate quantitatively the extent of the infarcted mass. Plasma trace element levels were measured in blood samples following centrifugation and storage at ?80°C. The main results were as followed: In G1 patients who have been subjected to thrombolysis, an important release of myosin was measured in plasma, with a peak at D6 (1678 vs 95 μU/L at H0). In those G1 patients after MI:

1. A significant increase in plasma copper levels was observed from day 4 to day 10 postinfarction (×1.15 in reference to the baseline data at H0);
2. A decrease in plasma zinc levels was observed and was maximum 12 h after the onset of the thrombolytic treatment;
3. A decrease in selenium concentration was observed in G1, as well as in G2 patients, compared to the control groups (80% of G3 and G4 values); and
4. A significant decrease in plasma iron levels was observed in G1 (67.8% of G3 and G4 control values) and was significant from H0 to day 7 (p<0.01).

In conclusion, this study underlined the time-course evolution of plasma trace element levels in the followup of patients who have been subjected to thrombolysis following a MI and the potential prognostic implication of such variations.     

Highest Plasma Phenylalanine Levels in (Very) Premature Infants on Intravenous Feeding; A Need for Concern

Objective

To analyse the association in newborns between blood levels of phenylalanine and feeding method and gestational age.

Study Design

This observational, cross-sectional study included a sample of 11,829 infants between 2008 and 2013 in a Spanish region. Data were recorded on phenylalanine values, feeding method [breast, formula, mixed (breast plus formula), or partial or fully intravenous feeding], gestational age in weeks (<32, 32–37, ≥37), gender and days since birth at the moment of blood collection. Outcomes were [phenylalanine] and [phenylalanine] ≥95th percentile. Associations were analysed using multivariate models [linear (means difference) and logistic regression (adjusted odds ratios)].

Results

Higher phenylalanine values were associated with lower gestational age (p<0.001) and with intravenous feeding (p<0.001).

Conclusion

The degree of prematurity and intravenous feeding influenced the plasma concentration of phenylalanine in the newborn. Caution should be taken in [phenylalanine] for newborns with intravenous feeding, monitoring them carefully. Very preterm infants given the recommended amount of amino acids should also be strictly monitored. These findings should be taken into consideration and call for adapting the amounts to the needs of the infant.     

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

Co-induction of sucrose transport and invertase activities in a thermophilic fungus,Thermomyces lanuginosus

The incorporation of sucrose into the thermophilic fungus,Thermomyces lanuginosus, occurred only in mycelia previously exposed to sucrose or raffinose. Sucrose uptake and invertase were inducible. Both activities appeared in sucrose-induced mycelia at about the same time. Both activities declined almost simultaneously following the exhaustion of sucrose in the medium. The sucrose-induced uptake system was specific for -fructofuranosides as revealed by competition with various sugars. The induction of sucrose uptake system was blocked by cycloheximide, showing that it was dependent on new protein synthesis. Transport of sucrose did not seem to be dependent on ATP. Rather, uptake of this sugar seemed to be driven by a proton gradient across the plasma membrane. The uptake system showed Michaelis-Menten kinetics.Abbreviations FCCP carbonylcyanide p-trifluoromethylphenyl hydrazone - 2,4-DNP 2,4-dinitrophenol     

Human amylin proteotoxicity impairs protein biosynthesis,and alters major cellular signaling pathways in the heart,brain and liver of humanized diabetic rat model in vivo

Introduction

Chronic hypersecretion of the 37 amino acid amylin is common in type 2 diabetics (T2D). Recent studies implicate human amylin aggregates cause proteotoxicity (cell death induced by misfolded proteins) in both the brain and the heart.

Objectives

Identify systemic mechanisms/markers by which human amylin associated with cardiac and brain defects might be identified.

Methods

We investigated the metabolic consequences of amyloidogenic and cytotoxic amylin oligomers in heart, brain, liver, and plasma using non-targeted metabolomics analysis in a rat model expressing pancreatic human amylin (HIP model).

Results

Four metabolites were significantly different in three or more of the four compartments (heart, brain, liver, and plasma) in HIP rats. When compared to a T2D rat model, HIP hearts uniquely had significant DECREASES in five amino acids (lysine, alanine, tyrosine, phenylalanine, serine), with phenylalanine decreased across all four tissues investigated, including plasma. In contrast, significantly INCREASED circulating phenylalanine is reported in diabetics in multiple recent studies.

Conclusion

DECREASED phenylalanine may serve as a unique marker of cardiac and brain dysfunction due to hyperamylinemia that can be differentiated from alterations in T2D in the plasma. While the deficiency in phenylalanine was seen across tissues including plasma and could be monitored, reduced tyrosine was seen only in the brain. The 50 % reduction in phenylalanine and tyrosine in HIP brains is significant given their role in supporting brain chemistry as a precursor for catecholamines (dopamine, norepinephrine, epinephrine), which may contribute to the increased morbidity and mortality in diabetics at a multi-system level beyond the effects on glucose metabolism.

     

Weight loss and weight maintenance obtained with or without GLP-1 analogue treatment decrease branched chain amino acid levels

Introduction

Increased levels of circulating branched chain amino acids (BCAAs), as well as phenylalanine, and tyrosine have been suggested to be involved in the pathogenesis of insulin resistance and type 2 diabetes. However, it is unknown how these metabolites are affected by weight loss, and during weight-maintaining treatment with glucagon-like peptide-1 receptor agonist (GLP-1 RA).

Objective

We aimed to characterize changes in metabolites related to protein turnover and glycolysis after a weight loss intervention followed by long term weight maintenance with/without GLP-1 RA.

Methods

Fifty-eight obese individuals underwent a diet-induced 12 % body weight loss during 8 weeks. Participants were randomized to weight maintenance with or without administration of the GLP-1 RA liraglutide (1.2 mg/day) for 52 weeks. Metabolomic profiling by high-throughput proton nuclear magnetic resonance spectroscopy was used for quantification of metabolites.

Results

The weight loss was maintained in both groups and was associated with 9–20 % decreases in plasma concentrations of alanine, phenylalanine, histidine, tyrosine and the BCAAs leucine, isoleucine and valine (p < 0.05). Plasma citrate levels increased during weight loss (p = 5.2 × 10^?15) and showed inverse correlation with insulin resistance measured by HOMA–IR levels (r = ?0.318, p = 0.025). Valine concentrations were lower in the control group compared to the GLP-1RA group during weight maintenance (p = 0.005).

Conclusion

Weight loss is associated with marked changes in plasma concentrations of eight amino acids and glycolysis-related metabolites. Levels of the suggested type 2 diabetes risk markers (BCAAs) remain low during long-term weight maintenance.

     

Protein Intake and Plasma Amino-acids of Infants of Low Birth Weight

The plasma amino-acid levels in infants of low birth weight fed on expressed human milk and on a proprietary breast-milk substitute, S26, with a protein intake of not more than 4·5 g/kg/day were compared with those in infants fed on an evaporated milk formula whose protein intake ranged from 6·15 to 12·3 g/kg/day, as well as with normal infants on normal feeds and protein intake. In general, there was little difference between the levels in infants of low birth weight and in normal infants on the same protein intake. The five infants of low birth weight on high protein intake had generally higher levels of plasma amino-acids compared with the group on the lower protein intake, and in particular the levels of tyrosine, phenylalanine, methionine, and cystathionine could be extremely high. Apart from methionine these high levels may be the result both of a reduction in activity of the enzymes involved in the metabolism of these amino-acids, due to the immaturity of the infant, and of the increased stress of a high protein intake. In view of a possible long-term effect of abnormally high plasma amino-acid levels it is suggested that the protein intake of infants of low birth weight should not exceed 6 g/kg/day.     

Phenylketonuria—The Guthrie Screening Test—A Method of Quantitation,Observations on Reliability and Suggestions for Improvement

The Guthrie bacterial inhibition assay method of screening neonatal infants for phenylketonuria (pku) initiated mass screening for inborn errors of metabolism. It is a simple, cheap procedure admirably suited to private local use as against private or public central use. However, using parallel fluorometric determinations as a basis for comparison, and 4 mg per 100 ml serum phenylalanine as a presumptive positive threshhold, the Guthrie test yielded 53 per cent “false negatives.” Extrapolating from a combination of our data and reported phenylalanine levels at three days of age or less in proved pku patients, it is estimated the Guthrie test might fail to detect one of 25 pku patients screened at three days of age or less. Means to diminish this risk are considered.     

Iron deficiency in the rat: biochemical studies of fetal metabolism

The effects of dietary-induced iron deficiency on fetal and maternal metabolism were studied in the rat. Concentrations of phenylalanine, but not tyrosine, were significantly elevated in plasma from iron-deficient maternal and fetal rats at day 20 of gestation with individual fetal plasma levels of phenylalanine as high as 10 mg per 100 ml. Concentrations of total 5-hydroxyindole compounds were significantly decreased in brain tissue from iron-deficient fetuses (day 20 of gestation), suggesting that synthesis of the compounds may be inhibited by iron deficiency. Mitochondrial NADH oxidase activity was markedly decreased (60%) in homogenates of fetuses at day 14 of gestation and may account for the high fetal resorption rate and small fetal size observed in the rat in iron deficiency.     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites (phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.     

Inhibition of brain and liver 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphate decarboxylase in experimental hyperphenylalaninemia

Experimental hyperphenylalaninemia has been induced in 5-day-old chicks by dietary treatments with phenylalanine and -methylphenylalanine. An increase of nearly 8-fold in plasma Phe/Tyr ratio was found after 4 days of supplementation the standard diet with 5% phenylalanine plus 0.4% -methylphenylalanine. The increase in this ratio was about 13-fold after 9 days of the same treatment. Similar results were observed in brain and liver, although the increases were smaller than those found in plasma. Total body, brain and liver weight decreased after 9 days of treatment. Phenylalanine plus -methylphenylalanine administration to 5-day-old chicks produced a significant decrease in the 3-hydroxy-3-methylglutary-CoA reductase and mevalonate-5-pyrophosphate decarboxylase specific activities from both brain and liver. These results demonstrated for the first time that experimental hyperphenylalaninemia inhibited different enzyme activites directly implicated in the regulation of cholesterogenesis. Therefore, a reduced cholesterol synthesis in brain may evidenciate the theory of an impaired myelination leading to mental retardation in phenylketonuria patients.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Ammonia-nitrogen production by the bivalve molluscTapes japonica and its recovery by the red seaweedHypnea musciformis in a tropical mariculture system

Production and recovery of ammonia-N was studied in the second and third trophic levels of a mariculture system on St. Croix, US Virgin Islands. The diatomChaetoceros curvisetus, grown on nutrients in artificially upwelled deep water from 870 m depth, was the food source forTapes japonica. Consumption of the phytoplankton byT. japonica increased throughout the day and decreased at night, and was related to corresponding changes in algal culture density. Feeding efficiency was highest at night. Ammonia-N production by the clams fluctuated over a typical 24 h period; dropping during the day and increasing at night. The ammonia-N concentration in the shellfish tank effluent was inversely related to the quantity of phytoplankton consumed by the clams. At all ration levels the small clams produced ammonia-N at a greater rate than large clams.H. musciformis fragmented and washed out of some tanks; the fragmentation was related to high ammonia-N concentrations in the inflowing water. High light intensity and temperature alone do not appear to cause fragmentation, but may have induced a trace nutrient deficiency inH. musciformis grown in the ammonia-rich seawater. Where fragmentation was not obvious, ammonia-N uptake per gH. musciformis was highly correlated with the average ammonia-N concentration of the inflowing seawater both day and night. Percent uptake of ammonia-N increased with increasing concentration of the nutrient in the inflowing seawater, reaching a plateau of about 70% uptake of the available ammonia-N at concentrations above 4 g-at/l.