Kegg Pathway: Urea cycle and metabolism of amino groups

Mol Genet Metab. 2008 Aug; 94(4): 397-402
Tuchman M, Lee B, Lichter-Konecki U, Summar ML, Yudkoff M, Cederbaum SD, Kerr DS, Diaz GA, Seashore MR, Lee HS, McCarter RJ, Krischer JP, Batshaw ML,

Inherited Urea cycle disorders comprise eight disorders (UCD), each caused by a deficiency of one of the proteins that is essential for Ureagenesis. We report on a cross-sectional investigation to determine clinical and laboratory characteristics of patients with UCD in the United States. The data used for the analysis was collected at the time of enrollment of individuals with inherited UCD into a longitudinal observation study. The study has been conducted by the Urea cycle Disorders Consortium within the Rare Diseases Clinical Research Network (RDCRN) funded by the National Institutes of Health. One-hundred eighty-three patients were enrolled into the study. Ornithine transcarbamylase (OTC) deficiency was the most frequent disorder (55%), followed by argininosuccinic aciduria (16%) and citrullinemia (14%). Seventy-nine percent of the participants were white (16% Latinos), and 6% were African American. Intellectual and developmental disabilities were reported in 39% with learning disabilities (35%) and half had abnormal neurological examination. Sixty-three percent were on a protein restricted diet, 37% were on Na-phenylbutyrate and 5% were on Na-benzoate. Forty-five percent of OTC deficient patients were on L-citrulline, while most patients with citrullinemia (58%) and argininosuccinic aciduria (79%) were on L-arginine. Plasma levels of branched-chain amino acids were reduced in patients treated with ammonia scavenger drugs. Plasma glutamine levels were higher in proximal UCD and in neonatal type disease. The RDCRN allows comprehensive analyses of rare inherited UCD, their frequencies and current medical practices.

J Anim Physiol Anim Nutr (Berl). 2008 Apr; 92(2): 149-56
Strieker MJ, Morris JG, Avery EH, Freedland RA, Rogers QR

Previous experiments have shown that increasing the dietary crude protein (CP) of cats does not increase Urea cycle enzymes or alanine amino transferase as occurs in rats. Also when an essential amino acid (EAA) is limiting in a diet for growing kittens, the kittens do not exhibit an amino acid imbalance when other EAAs are added to the diet. To study the metabolic basis for these observations which are different from that found in omnivores and herbivores, the hypothesis that increased dietary CP decreases methionine catabolism, so more is spared for growth, was tested. Fifteen male kittens were randomly assigned to one of three dietary treatments. Each diet contained 2.5 g l-methionine/kg diet and 200, 300 or 500 g CP/kg diet. The livers and kidneys were removed and assayed for methionine transaminase (MTA), cystathionase (CASE) and cystathionine synthase (CS). Free amino acid concentrations were determined in liver, kidney and plasma. The 300 and 500 g CP/kg groups had significantly greater kidney weights and body weight gains than the 200 g CP/kg group. Hepatic MTA activity was lower in the 300 than the 200 or 500 g CP/kg groups (p < 0.05). Renal MTA and CASE activities were 35% and 50% greater, respectively, for the 500 g CP/kg group than for the 200 g CP/kg diet group (p < 0.05). Renal CS activities for the 300 and 500 g CP/kg groups were 29% (p > 0.05) and 38% (p < 0.05) greater, respectively, than the 200 g CP/kg group. Cyst(e)ine concentrations were lower in the livers of the 500 g CP/kg group than the 200 g CP/kg group (p < 0.05). Cystathionine was lower in plasma and kidney from the 500 g CP/kg diet group than from the 200 g CP/kg diet group (p < 0.05). It was concluded that the metabolic basis for the increased growth of kittens fed diets marginally limiting in methionine, with increasing concentrations of dietary CP, was not mediated through decreased enzyme activity associated with the catabolism of methionine, but was the result of an increase in food (methionine) intake.

J Inherit Metab Dis. 2007 Aug; 30(4): 515-21
Auray-Blais C, Cyr D, Drouin R

The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of more than 2,500,000 newborns in the province of Quebec for 25 inherited Mendelian disorders divided into two groups. The first group concerns Urea cycle disorders (citrullinaemia, hyperargininaemia, argininosuccinic aciduria), ketotic hyperglycinaemia, and organic acidurias (methylmalonic aciduria, glutaric aciduria type I, etc.); the second group relates to disorders of amino acid metabolism (cystathioninuria, prolidase deficiency, etc.) and transport (Fanconi syndrome, cystinurias, Hartnup syndrome, etc.). The main goal of the Programme is to detect and prevent these genetic diseases, some detectable only in urine, before the onset of clinical symptoms. A multiplex thin-layer chromatography methodology was developed, in which metabolites in urine are resolved and visualized by the sequential application of four different reagents to detect aminoacidopathies and organic acidurias. The technique is simple, reproducible, inexpensive and rapid, allowing the analysis of 500 samples daily by a single technician. The voluntary compliance of the parents is excellent, averaging 90% per year. Over the years, we have established a dynamic process, developing techniques or new reagents to detect as many treatable disorders as possible, now evaluating macromolecules associated with lysosomal storage disorders, mainly globotriaosylceramide (Gb3) for Fabry disease. We present here the methodology, infrastructure in place, results and recent statistics of the well-established Quebec Mass Urinary Screening Programme. We also report a study by tandem mass spectrometric analysis of urinary Gb3 in Fabry disease for the follow-up and monitoring of Fabry patients, as well as for its possible application to mass and high-risk screening programmes.

Anal Chem. 2007 Jan 1; 79(1): 89-97
Gu H, Chen H, Pan Z, Jackson AU, Talaty N, Xi B, Kissinger C, Duda C, Mann D, Raftery D, Cooks RG

The effect of diet on metabolites found in rat urine samples has been investigated using nuclear magnetic resonance (NMR) and a new ambient ionization mass spectrometry experiment, extractive electrospray ionization mass spectrometry (EESI-MS). Urine samples from rats with three different dietary regimens were readily distinguished using multivariate statistical analysis on metabolites detected by NMR and MS. To observe the effect of diet on metabolic pathways, metabolites related to specific pathways were also investigated using multivariate statistical analysis. Discrimination is increased by making observations on restricted compound sets. Changes in diet at 24-h intervals led to predictable changes in the spectral data. Principal component analysis was used to separate the rats into groups according to their different dietary regimens using the full NMR, EESI-MS data or restricted sets of peaks in the mass spectra corresponding only to metabolites found in the Urea cycle and metabolism of amino groups pathway. By contrast, multivariate analysis of variance from the score plots showed that metabolites of purine metabolism obscure the classification relative to the full metabolite set. These results suggest that it may be possible to reduce the number of statistical variables used by monitoring the biochemical variability of particular pathways. It should also be possible by this procedure to reduce the effect of diet in the biofluid samples for such purposes as disease detection.

Alcohol Alcohol. 2006 Jul-Aug; 41(4): 372-8
Hasselblatt M, Krampe H, Jacobs S, Sindram H, Armstrong VW, Hecker M, Ehrenreich H

AIMS: Data on recovery from hormonal and metabolic sequelae of alcoholism in strictly controlled alcohol abstinence are mainly restricted to short-term abstention. Our previous findings of persistently decreased plasma and urinary Urea concentrations in long-term abstinent alcoholics prompted us to further elucidate this unexplained phenomenon. METHODS: The response of circulating Urea cycle metabolites and glucose-regulating hormones to an intravenous load (30 g) of arginine hydrochloride was investigated in abstinent male alcoholics (n = 14) after complete recovery of all routine liver parameters and compared with that in healthy male controls (n = 15). RESULTS: The arginine challenge provoked (i) higher peak concentrations of arginine and increased arginine/ornithine and ornithine/citrulline ratios in the plasma of abstinent alcoholics; (ii) augmented plasma glutamine concentrations in alcoholics in the presence of comparable levels in both experimental groups of plasma glutamate, ammonia, and nitrate/nitrite; (iii) parallel increases in plasma Urea concentrations over the respective baseline levels but distinctly higher urinary Urea excretion in controls; (iv) a blunted blood glucose response to arginine in alcoholics together with a reduced insulin and glucagon surge; and (v) an elevated growth hormone peak as compared with controls. CONCLUSIONS: Application of an intravenous arginine challenge reveals profound and lasting metabolic and hormonal disturbances in abstinent alcoholics, affecting Urea cycle and gluconeogenesis. The common denominator of many of these changes may be an acquired irreversible deficiency in cellular energy regulation.

Clin Chim Acta. 2006 Feb; 364(1-2): 209-16
Chuang CK, Lin SP, Chen HH, Chen YC, Wang TJ, Shieh WH, Wu CJ

BACKGROUND: The high prevalence of protein-energy malnutrition is a critical issue for patients with end stage renal disease (ESRD) on hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Levels of plasma and intracellular amino acids are significant indicators of protein metabolism and nutritional status assessment. We measured plasma FAAs in patients on maintenance dialysis and to provide information in monitoring the therapeutic strategy, particularly in AA supplementary therapy or protein restriction. METHODS: Fifty-five patients with ESRD were investigated, 25 on HD (male : female=14 : 11; 48-67 y) and 30 on CAPD (male : female=17 : 13; 45-64 y). The subjects had been on dialysis for an average of 13 months (range, 9 to 22 months). Their plasma FAAs (including their intermediate metabolites) were measured by ion exchange chromatography before and after HD or during CAPD and were compared with data obtained from 20 age- and sex-matched healthy controls. RESULTS: The total plasma FAA levels (Urea and free ammonia, NH3 were excluded) in pre-HD samples (3911 +/- 709 micromol/l) was significantly higher than in the other groups (2570 +/- 378 in control, 3210 +/- 640 in post-HD, and 3468 +/- 271 in CAPD samples). The mean plasma FAA concentrations differed significantly between pre-HD and controls and between pre-HD and CAPD samples (p<0.05). No significant differences were found among the other group comparisons. Comparing individual FAA concentrations, only citrulline differed significantly among all groups (p<0.05), whereas serine, glutamine, beta-alanine, beta-aminoisobutyric acid, and gamma-aminobutyric acid were not different. Concentrations of some FAAs involved in the Urea cycle, e.g., arginine, aspartic acid, citrulline, and ornithines, and solutes Urea and NH3, were significantly increased. Ratios of tyrosine/phenylalanine and valine/glycine ratios were markedly reduced in all patients on dialysis compared with controls. CONCLUSION: FAAs either from dietary uptake or protein catabolism are substantially retained in the plasma of patients with ESRD, possibly producing higher levels of the waste products (Urea and NH3) through the Urea cycle and ammonia metabolism in liver. Maintenance dialysis can effectively eliminate excess FAAs in plasma, as there was a 17.9% reduction post-HD. The abnormalities in FAA metabolism found in patients with ESRD necessitate careful consideration of dialysis and dietary measures.

amino Acids. 2005 May; 28(3): 297-303
Tujioka K, Lyou S, Fukaya Y, Sano A, Hayase K, Yokogoshi H

We have shown that urinary Urea excretion increased in rats fed a low quality protein. The purpose of present study was to determine whether an addition of dietary limiting amino acids affected Urea synthesis in rats fed a low gluten diet. Experiments were done on three groups of rats given diets containing 10% gluten, 10% gluten +0.5% L-lysine or 10% gluten+0.5% L-lysine, 0.2% L-threonine and 0.2% L-methionine for 10 d. The urinary excretion of Urea, and the liver concentrations of serine and ornithine decreased with the addition of dietary L-lysine, L-threonine and L-methionine. The fractional and absolute rates of protein synthesis in tissues increased with the treatment of limiting amino acids. The activities of hepatic Urea-cycle enzymes was not related to the Urea excretion. These results suggest that the addition of limiting amino acids for the low gluten diet controls the protein synthesis in tissues and hepatic ornithine and decline Urea synthesis.

Mol Biol Evol. 2005 Jan; 22(1): 1-11
Cunchillos C, Lecointre G

The darwinian concept of "descent with modification" applies to metabolic pathways: pathways sharing similarities must have inherited them from an exclusive, hypothetical ancestral pathway. Comparative anatomy of biochemical pathways is performed using five criteria of homology. Primary homologies of "type I" were defined as several pathways sharing the same enzyme with high specificity for its substrate. Primary homologies of "type II" were defined as the sharing of similar enzymatic functions, cofactors, functional family, or recurrence of a set of reactions. Standard cladistic analysis is used to infer the evolutionary history of metabolic development and the relative ordering of biochemical reactions through time, from a single matrix integrating the whole basic universal metabolism. The cladogram shows that the earliest pathways to emerge are metabolism of amino acids of groups I and II (Asp, Asn, Glu, and Gln). The earliest enzymatic functions are mostly linked to amino acid catabolism: deamination, transamination, and decarboxylation. For some amino acids, catabolism and biosynthesis occur at the same time (Asp, Glu, Lys, and Met). Catabolism precedes anabolism for Asn, Gln, Arg, Trp, His, Tyr, and Phe, and anabolism precedes catabolism for Pro, Ala, Leu, Val, Ile, Cys, Gly, Ser, and Thr. The Urea cycle evolves from arginine synthesis. metabolism of fatty acids and sugars develops after the full development of metabolism of amino acids of groups I and II, and they are associated with the anabolism of amino acids of groups III and IV. Syntheses of aromatic amino acids are branched within sugar metabolism. The Krebs cycle occurs relatively late after the setting of metabolism of amino acids of groups I and II. One portion of the Krebs cycle has a catabolic origin, whereas the other portion has an anabolic origin in pathways of amino acids of groups III and IV. It is not possible to order glycolysis and gluconeogenesis with regard to the Krebs cycle, as they all belong to "period 6." Pentose-phosphate and Calvin cycles are later (periods 7 and 8, respectively). Cladistic analysis of the structure of biochemical pathways makes hypotheses in biochemical evolution explicit and parsimonious.

J Dairy Sci. 2004 May; 87(5): 1325-35
Velez JC, Donkin SS

Somatotropin (ST) increases milk production and through coordinated changes in hepatic glucose synthesis and amino acid metabolism in dairy cows. The objective of this study was to determine the effects of ST on hepatic mRNA expression for phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC), enzymes that are critical to the synthesis of glucose in liver and hepatic mRNA expression for carbamylphosphate synthetase I (CPS-I), argininosuccinate synthetase (AS), and ornithine transcarbamylase (OTC), critical enzymes of the Urea cycle. Eighteen cows were randomly allocated to 2 treatment groups and received either recombinant bovine ST (Posilac; Monsanto, St. Louis, MO) or saline injections at 14-d intervals during a 42-d period. Expression of mRNA was determined using Northern blot analysis. Nuclei, isolated from liver biopsy samples, were used to determine effects of ST on transcription rate of PEPCK. Milk production was increased with ST (37.3 vs. 35.1+/-0.6 kg/ d). Plasma NEFA was increased with ST (299 vs. 156+/-34 microM). There were no differences in the expression of CPS-I, AS, and OTC mRNA with ST. Expression of PEPCK and IGF-I mRNA were increased with ST but PC mRNA was unchanged. The data indicate increased PEPCK mRNA in cows given ST and indicates a greater capacity for gluconeogenesis from gluconeogenic precursors that form oxaloacetate. The effects of ST to elevate PEPCK mRNA expression require chronic administration and involve increased transcription of the PEPCK gene.

J Nutr Sci Vitaminol (Tokyo). 2003 Aug; 49(4): 247-55
Imai S, Fujita K, Miura M, Saeki T, Kotaru M, Iwami K

The activity of hepatic serine dehydratase (SDH) increases in tandem with its gene expression when the intake of protein greatly exceeds protein requirements. The actual conditions of plasma free amino acids and pancreatic hormones in weanling and mature rats when fed SDH-inducible and non-inducible diets were examined in relation to incentive factors to secure high SDH activity from a physiological standpoint. Both weanling and mature groups differing in protein requirements were allowed free access to respective diets diverse in protein content (i.e. 25% or 50% casein diet for the former and 6% or 25% casein diet for the latter) during the dark cycle (lights-out) over a period of 1 wk. Despite the difference in protein intake among these groups, there were no conspicuous changes in the plasma concentration of the Urea or total or essential amino acids. Therefore, it appears that the individual amino acids did not up regulate the gene and function expressions of SDH merely by their superabundance and subsequent disposal. Portal venous insulin concentration was far higher in mature groups than in weanling groups, although there was little difference between the two groups of the same age in terms of insulin or glucagon concentration and their ratio in abdominal vena cava blood. Accordingly, it follows that the SDH gene undergoes transcriptional regulation through a combined signaling pathway triggered by perceiving surplus protein nutrition as a whole rather than directly through already-known plasma constituents such as free amino acids or pancreatic hormones in the circulatory system.

Biotechnol Bioeng. 2003 Aug 20; 83(4): 400-15
Lee K, Berthiaume F, Stephanopoulos GN, Yarmush ML

The liver plays an important role in the overall negative nitrogen balance leading to muscle wasting commonly observed in patients following many conditions, including severe injury, cancer, and diabetes. In order to study changes in liver metabolism during the establishment of such catabolic states, we used a rat skin burn injury model that induces hypermetabolism and muscle wasting. At various times during the first week following the injury, livers were isolated and perfused in a recirculating system under well-defined conditions. We applied a steady-state metabolic flux analysis model of liver metabolism and then used k-means clustering to objectively group together reaction flux time profiles. We identified six distinct groups of reactions that were differentially responsive: (1) pentose phosphate pathway (PPP); (2) amino acid oxidation reactions leading to the formation of tricarboxylic acid (TCA) cycle intermediates; (3) gluconeogenesis; (4) TCA-cycle and mitochondrial oxidation; (5) lipolysis, beta-oxidation, and ketone body formation; and (6) Urea-cycle. Burn injury sequentially upregulated the Urea-cycle, the PPP, and the TCA-cycle, in order, while beta-oxidation and gluconeogenesis remained unchanged. The upregulation of the PPP was transient, whereas the rise in Urea- and TCA-cycle fluxes was sustained. An ATP balance predicted an increased production of ATP and energy expenditure starting on day 3 post-burn, which correlated with the induction of the oxidative phosphorylation uncoupler uncoupling protein-2. We conclude that metabolic profiling using flux analysis and clustering analysis is a useful methodology to characterize the differential activation of metabolic pathways in perfused organs and to identify specific key pathways that are sensitive to a stimulus or insult without making a priori assumptions.

Clin Nutr. 2003 Feb; 22(1): 93-8
Pita AM, Wakabayashi Y, Fernandez-Bustos MA, Virgili N, Riudor E, Soler J, Farriol M

BACKGROUND AND AIMS: The small intestine contains several enzymes involved in arginine synthesis and converts glutamine to citrulline, the major compound for endogenous arginine synthesis. This study was conducted to assess the plasma status of Urea-cycle intermediates and orotic urinary excretion in short-bowel patients. METHODS:Thirteen stable short-bowel syndrome patients (7 men; 60.2+/-15.2 years) were studied. Patients were divided into moderately resected (Group A; n=6) and severely resected (Group B; n=7) according to their remnant bowel length (Group A: 61-150 cm; Group B: < or =60 cm). All subjects were consuming an oral diet plus dietetic supplements. Plasma Urea-cycle amino acids, ammonium and urinary orotic acid were determined. RESULTS: Plasma glutamine levels were significantly higher in both patient groups than in the control group (P<0.001). Regarding citrulline, Group B levels were significantly lower vs. controls (P<0.001). Comparisons between patient groups showed higher arginine in Group A (P<0.05) and non-statistically lower citrulline in Group B. Blood ammonium and orotic urinary excretion were normal. CONCLUSIONS: Although plasma citrulline and glutamine alterations were found, patients showed no hyperammonemia or orotic aciduria, which suggests a certain degree of adaptation in arginine and related amino acid metabolism, when an adequate dietary supply of arginine is provided.

Nucleic Acids Res. 2002 Nov 1; 30(21): 4728-39
Tanaka H, Ryu GH, Seo YS, Tanaka K, Okayama H, MacNeill SA, Yuasa Y

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the completion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.

Biosystems. 2002 Feb; 65(1): 61-78
Kohn MC, Tohmaz AS, Giroux KJ, Blumenthal GM, Feezor MD, Millington DS

Urea production in human liver was described by a MetaNet graph, a flowchart-like representation of metabolic pathways that includes parameters for the kinetic constants of the constituent enzymes. Formal operations on the graph facilitate the identification of ligand-binding equilibria that participate in feedback regulation in the network of biochemical reactions. The state of the biochemical network is specified by the concentrations of the intermediates. At any particular time, the influence of an identified locus of regulation is proportional to the respective fractional saturation of the corresponding binding site. Enzymes that make or consume the feedback chemicals share in the control of the strength of the feedback signal in proportion to their fractional saturation. This model predicts control of Urea production by the processes that deliver amino groups to the Urea cycle enzymes more than by the cycle enzymes themselves. Mitochondrial membrane transport processes are important for transmission of information through the network, but irreversible enzymes and processes far from equilibrium control the strength of the feedback signal. Systematic variation of the parameter values by amounts comparable to the expected variability of their measured values indicated a high probability of invariance in the identities of the predicted control points. The properties of the model are consistent with those of error-tolerant scale-free networks. These results demonstrate the robustness of a MetaNet model's predictions with respect to uncertainties in the values of its parameters.

J Med Chem. 2001 Dec 20; 44(26): 4628-40
Honma T, Yoshizumi T, Hashimoto N, Hayashi K, Kawanishi N, Fukasawa K, Takaki T, Ikeura C, Ikuta M, Suzuki-Takahashi I, Hayama T, Nishimura S, Morishima H

Identification of a selective inhibitor for a particular protein kinase without inhibition of other kinases is critical for use as a biological tool or drug. However, this is very difficult because there are hundreds of homologous kinases and their kinase domains including the ATP binding pocket have a common folding pattern. To address this issue, we applied the following structure-based approach for designing selective Cdk4 inhibitors: (1) identification of specifically altered amino acid residues around the ATP binding pocket in Cdk4 by comparison of 390 representative kinases, (2) prediction of appropriate positions to introduce substituents in lead compounds based on the locations of the altered amino acid residues and the binding modes of lead compounds, and (3) library design to interact with the altered amino acid residues supported by de novo design programs. Accordingly, Asp99, Thr102, and Gln98 of Cdk4, which are located in the p16 binding region, were selected as first target residues for specific interactions with Cdk4. Subsequently, the 5-position of the pyrazole ring in the pyrazol-3-ylUrea class of lead compound (2a) was predicted to be a suitable position to introduce substituents. We then designed a chemical library of pyrazol-3-ylUrea substituted with alkylaminomethyl groups based on the output structures of de novo design programs. Thus we identified a highly selective and potent Cdk4 inhibitor, 15b, substituted with a 5-chloroindan-2-ylaminomethyl group. Compound 15b showed higher selectivity on Cdk4 over those on not only Cdk1/2 (780-fold/190-fold) but also many other kinases (>430-fold) that have been tested thus far. The structural basis for Cdk4 selective inhibition by 15b was analyzed by combining molecular modeling and the X-ray analysis of the Cdk4 mimic Cdk2-inhibitor complex. The results suggest that the hydrogen bond with the carboxyl group of Asp99 and hydrophobic van der Waals contact with the side chains of Thr102 and Gln98 are important. Compound 15b was found to cause cell cycle arrest of the Rb(+) cancer cell line in the G(1) phase, indicating that it is a good biological tool.

J Hepatol. 2001 Dec; 35(6): 700-6
Grøfte T, Jensen DS, Greisen J, Tygstrup N, Vilstrup H

Background/Aims: Long-term steroid treatment causes protein wasting. Liver contributes towards this by upregulating Ureagenesis. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are anabolic agents with specific hepatic effects. It is unknown whether IGF-I alone and/or in combination with GH have any effect on established hepatic amino-N catabolism during long-term glucocorticoid treatment.Methods: We measured the spontaneous (UNSR) and the substrate standardized rate of Urea nitrogen synthesis (STUNSR), N-balance and mRNA levels of Urea cycle enzymes in controls (placebo) and four longterm steroid treated groups given (1) prednisolone 4 mg/kg/day during 28 days (St) (2) +GH 1 mg/kg/day from day 21-28 (StGH) (3) +IGF-I 1.5 mg/kg/day 21-28 (StIGF) (4) GH +IGF-I (StGHIGF).Results: Steroid induced weight loss was stepwisely reversed by IGF-I, GH and both. UNSR, STUNSR and mRNA levels of Urea cycle enzymes in the liver increased markedly after steroid treatment, and was normalized after co-administration of GH and IGF-I. N-balance improved after GH and IGF-I administration.Conclusions: Our results expands the knowledge of beneficial effects of GH on short-term steroid catabolism to include effects of IGF-I and IGF-I combined with GH on long-term steroid catabolism. Both peptides prevent steroid induced hepatic protein wasting and thereby contribute towards whole body anabolism. The effect in vivo is probably due to an effect of the peptides on Urea cycle enzyme mRNA.

Nucleic Acids Res. 2000 Nov 1; 28(21): 4340-9
Hang H, Rauth SJ, Hopkins KM, Lieberman HB

Schizosaccharomyces pombe rad9 mutations can render cells sensitive to hydroxyUrea (HU), gamma-rays and UV light and eliminate associated checkpoint controls. In vitro mutagenesis was performed on S.pombe rad9 and altered alleles were transplaced into the genome to ascertain the functional significance of five groups of evolutionarily conserved amino acids. Most targeted regions were changed to alanines, whereas rad9-S3 encodes a protein devoid of 22 amino acids normally present in yeast but absent from mammalian Rad9 proteins. We examined whether these rad9 alleles confer radiation and HU sensitivity and whether the sensitivities correlate with checkpoint control deficiencies. One rad9 mutant allele was fully active, whereas four others demonstrated partial loss of function. rad9-S1, which contains alterations in a BH3-like domain, conferred HU resistance but increased sensitivity to gamma-rays and UV light, without affecting checkpoint controls. rad9-S2 reduced gamma-ray sensitivity marginally, without altering other phenotypes. Two alleles, rad9-S4 and rad9-S5, reduced HU sensitivity, radiosensitivity and caused aberrant checkpoint function. HU-induced checkpoint control could not be uncoupled from drug resistance. These results establish unique as well as overlapping functional domains within Rad9p and provide evidence that requirements of the protein for promoting resistance to radiation and HU are not identical.

Proc Natl Acad Sci U S A. 2000 May 23; 97(11): 5773-8
Webb GC, Akbar MS, Zhao C, Steiner DF

Pancreatic beta cells respond to changes in blood glucose by secreting insulin and increasing insulin synthesis. To identify genes used in these responses, we have carried out expression profiling of beta cells exposed to high (25 mM) or low (5.5 mM) glucose by using oligonucleotide microarrays. Functional clustering of genes that averaged a 2.2-fold or greater change revealed large groups of secretory pathway components, enzymes of intermediary metabolism, cell-signaling components, and transcription factors. Many secretory pathway genes were up-regulated in high glucose, including seven members of the endoplasmic reticulum (ER) translocon. In agreement with array analysis, protein levels of translocon components were increased by high glucose. Most dramatically, the alpha subunit of the signal recognition particle receptor was increased over 20-fold. These data indicate that the translocon and ribosome docking are major regulatory targets of glucose in the beta cell. Analysis of genes encoding enzymes of intermediary metabolism indicated that low glucose brought about greater utilization of amino acids as an energy source. This conclusion was supported by observations of increased Urea production under low-glucose conditions. The above results demonstrate genome-wide integration of beta-cell functions at the level of transcript abundance and validate the efficacy of expression profiling in identifying genes involved in the beta-cell glucose response.

Pediatrics. 2000 Jan; 105(1): e10
Applegarth DA, Toone JR, Lowry RB

OBJECTIVE: To determine how many children with specific types of inborn errors of metabolism are born each year in British Columbia, Canada. This population provides a relatively unique setting for collection of accurate and uniform incidence data because the diagnoses are all made through one laboratory in a population with universal access to government-funded medical care. METHODOLOGY: We used the records of the Biochemical Diseases Laboratory, Children's Hospital, Vancouver (the central referral point for all metabolic diagnoses in British Columbia) to identify all patients diagnosed with the metabolic diseases defined below. We obtained incidence figures by including only the children diagnosed with the diseases covered in this article who were confirmed as having been born within the province for the years 1969 to 1996. The diseases covered were diseases of amino acids, organic acids, the Urea cycle, galactosemia, primary lactic acidoses, glycogen storage diseases, lysosomal storage diseases, and diseases involving specifically peroxisomal and mitochondrial respiratory chain dysfunction. Because the technology needed for diagnosis of specific disease groups was in place at different times our data for the different disease groups correspond to different time frames. We have also adjusted the time frames used to allow for the likelihood that some diseases may not come to medical attention for some time after birth. For instance the incidence of amino acid diseases was assessed throughout the whole of this time frame but the incidence of peroxisomal diseases was restricted to 1984 to 1996 because this was the time frame during which the technology needed for diagnosis was in place and reliable. Most disease group statistics included at least 400 000 births. RESULTS: The overall minimum incidence of the metabolic diseases surveyed in children born in British Columbia is approximately 40 cases per 100 000 live births. This includes phenylketonuria (PKU) and galactosemia which are detected by a newborn screening program. Metabolic diseases, which were not screened for at birth, ie, those with PKU and galactosemia subtracted from the total, have a minimal incidence of approximately 30 cases per 100 000 live births. This diagnostic dilemma group would present to pediatricians for diagnosis. Not all metabolic diseases have been surveyed and our data are restricted to the following metabolic disease groups. Approximately 24 children per 100 000 births (approximately 60% of the total disease groups surveyed) have a disease involving amino acids (including PKU), organic acids, primary lactic acidosis, galactosemia, or a Urea cycle disease. These children all have metabolic diseases involving small molecules. Approximately 2.3 children per 100 000 births ( approximately 5%) have some form of glycogen storage disease. Approximately 8 per 100 000 births (20%) have a lysosomal storage disease; approximately 3 per 100 000 births (7%-8%) have a respiratory chain-based, mitochondrial disease and approximately 3 to 4 per 100 000 (7%-8%) of births have a peroxisomal disease. The diseases involving subcellular organelles represent approximately half of the diagnostic dilemma group. The incidence of each of the specific diseases diagnosed, including apparently rare diseases such as nonketotic hyperglycinemia, is to be found in the text. The metabolic diseases reported in this survey represent over 10% of the total number of single gene disorders in our population. CONCLUSIONS: Our data provide a good estimate of metabolic disease incidence, for the disease groups surveyed, in a predominantly Caucasian population. Incidence data for metabolic diseases are hard to collect because in very few centers are diagnoses centralized for a population with uniform access to modern health care and this has been the case for our population during the course of the study. (ABSTRACT TRUNCATED)

Przegl Lek. 1998; 55(6): 337-41
Bik-Multanowski M

Inherited hyperammonemia disorders are caused by specific enzymatic defects in the Urea cycle or in metabolic pathways related to it. These disorders can be divided into the following groups: deficiencies of Urea cycle enzymes, transport defects of dibasic amino acids, organic acidemias, defects in beta-oxidation of fatty acids, transient hyperammonemia of the newborn-probably a not genetically determined disorder. Manifestation of the mentioned disorders includes elevated serum ammonia level resulting in altered level of consciousness and/or persisted vomiting. Occurrence of irreversible neurologic sequelae depends mostly on the extent of hyperammonemic period. Differential diagnosis includes blood gas, anion gap, plasma amino acids analysis and urine organic acids analysis. In some cases specific tissue enzymes activity measurement is necessary. Dialysis, sodium benzoate, sodium phenylacetate and arginine are used in the treatment of acute hyperammonemia. In addition oral or rectal neomycin and/or lactulose can be used, which reduces intestinal ammonia production.