Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo[4,5-f]quinoline bioactivation to mutagenic metabolites: significance of cytochrome P450 peroxygenase

We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to genotoxic metabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants. This increase was inhibited, in a concentration-dependent manner, by alpha-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) = 0.1 mM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'-deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway.     

The detection of cytochrome P450 2E1 and its catalytic activity in rat testis

The aim of this study was to compare the efficacy of 100 micrograms of salbutamol inhaled from a new metered-dose powder inhaler (MDPI, Leiras Taifun, Finland) with that of a same dose of salbutamol inhaled from a conventional pressurized metered-dose inhaler with a large volume spacer (pMDI + S) in protecting against methacholine (Mch) induced bronchoconstriction. This was a 3 day, randomized, cross-over, partly blinded, placebo-controlled multicentre study where the pMDI + S was used as an open control. Twenty-six asthmatic outpatients with a baseline FEV1 > or = 60% of predicted and with bronchial hyperreactivity (PD20 FEV1 < or = 890 micrograms of Mch) were studied. On each study day the patients underwent an Mch provocation 30 min after inhaling placebo from the MDPI or a dose of 100 micrograms of salbutamol from the MDPI and from the pMDI + S. PD20 FEV1 and dose-response slope [DRS; maximal change in FEV1 (%)/dose of Mch (mumol)] were used to evaluate efficacy. The median values of PD20 FEV1 were 250, 622 and 1737 micrograms after placebo MDPI, salbutamol pMDI + S and salbutamol MDPI, respectively. The corresponding DRS values were -11.0%, -4.5% and -2.0% mumol-1. With both parameters, all differences were statistically significant (P < 0.05). In conclusion, 100 micrograms of salbutamol inhaled from Leiras Taifun MDPI offers better protection against Mch-induced bronchoconstriction than 100 micrograms of salbutamol from a pMDI connected to a large volume spacer device.     

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.     

Trimethadione metabolism by human liver cytochrome P450: evidence for the involvement of CYP2E1

1. Caucasian liver samples were used in this study. N-demethylation of trimethadione (TMO) to dimethadione (DMO) was monitored in the presence of chemical inhibitors of CYPs, such as fluconazole, quinidine, dimethyl-nitrosamine, acetaminophen, phenacetin, chlorzoxazone and mephenytoin. Trimethadione N-demethylation was selectively inhibited by dimethylnitrosamine and chlorzoxazone (> 50%) and weakly inhibited by tolbutamide (12%) and fluconazole (22%), whereas other inhibitors showed no effect. This result suggested that TMO metabolism to DMO is mainly mediated by CYP2E1 and marginally by CYP2C and CYP3A4. 2. Fifteen human livers were screened and interindividual variability of TMO N-demethylation activity was 3-fold. Chlorzoxazone 6-hydroxylation activity was also measured and both activities were significantly correlated (r=0.735, p < 0.01). 3. DMO production by human cDNA expressed CYP enzymes was observed mainly for CYP2E1 (10.8 nmol/tube), marginally for CYP2C8 (0.22 nmol/tube) and not detectable for other CYP enzymes. 4. These results indicate that TMO metabolism is primarily catalysed by CYP2E1 and that trimethadione would be a suitable selective probe drug for the estimation of human CYP2E1 activity in vivo.     

Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain

Previous biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a 3H-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.     

Regional decreases in alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) and 6-[3H]cyano-7-nitroquinoxaline-2,3-dione ([3H]CNQX) binding in response to chronic low-level lead exposure: reversal versus potentiation by chronic dopamine agonist treatment

This study evaluated the hypotheses that in vivo lead (Pb) exposure would alter alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor binding and, based on known glutamate-dopamine interactions and Pb-induced changes in dopamine (DA) systems, that AMPA binding might be differentially influenced by DA agonist treatment under conditions of Pb exposure. Alterations in high-affinity ([3H]AMPA) versus total AMPA [6-[3H]cyano-7-nitroquinoxaline-2,3-dione ([3H]CNQX)] receptor binding were determined in medial frontal cortex, dorsal striatum, and nucleus accumbens of rats exposed to 0, 50, or 150 ppm of Pb acetate for 2 weeks or 8 months. Additional 8-month groups received chronic intermittent treatment with saline, the D1 agonist SKF82958, or the general DA agonist apomorphine. Two-week exposures increased AMPA receptor densities, whereas robust decreases occurred after 8 months of Pb; at the latter time point changes were more pronounced for high-affinity than total AMPA receptor binding, with high-affinity effects expressed preferentially in dorsal striatum and nucleus accumbens. DA agonist treatments almost fully reversed Pb-related declines in [3H]AMPA binding but either had no effect (apomorphine) or even further potentiated (SKF82958) the decreases in [3H]CNQX binding. One possible basis for the long-term (8-month) decrease in AMPA binding is a postsynaptic glutamatergic stimulation of non-NMDA receptors.     

Induction of cytochromes P450 2B and 2E1 in rat liver by isomeric picoline N-oxides

Pyridine derivatives are widely used solvents and precursors for the synthesis of chemicals of industrial importance. Oxidized metabolites have been implicated in the observed toxicity of pyridines and are known to induce drug-metabolizing enzymes in rat liver. In this study the three isomeric picoline (methylpyridine) N-oxides, as major oxidized metabolites of 2-, 3- and 4-picoline, were evaluated as inducers of cytochrome P450 (CYP) enzymes in rat liver. After a single dose of 100 mg/kg 24 h before sacrifice the 3- and 4-isomers were effective inducers of microsomal substrate oxidations associated with the phenobarbital-inducible CYPs 2B; upregulation of CYP2B protein was confirmed by immunoblotting. In contrast, the 2-isomer did not increase CYP2B protein or activity in rat liver but CYP2E1 protein expression was upregulated by the isomers to 160-200% of control. The three chemicals increased aniline 4-hydroxylation activity in rat liver, which is consistent with induction of CYPs 2B or 2E1 and 4-nitrophenol 2-hydroxylation activity was increased in microsomal fractions from 3- and 4-picoline N-oxide-treated rats. The activities of several other CYPs were also determined and CYP1A-dependent 7-ethylresorufin O-deethylation was increased (to approximately 6- and 2-fold of control) by the 3- and 4-isomer, respectively, whereas the activity of CYP3A-mediated androstenedione 6beta-hydroxylation was decreased by the agents--most notably by the 2-isomer. During NADPH-supported oxidation of CCl4, lipid peroxidation was increased in microsomes from 3- and 4-picoline N-oxide-pretreated rats and was modulated in vitro by the CYP2B inhibitor orphenadrine, but not by the CYP2E1 inhibitor 4-methylpyrazole. These findings establish that particular isomers of picoline N-oxide rapidly upregulate CYP2B or, to a lesser extent, CYP2E1 and implicate CYP2B in the enhanced lipid peroxidation observed in microsomes from rats treated with 3- and 4-picoline N-oxides. Such induction process may contribute to the hepatotoxicity of pyridines by enhancing the capacity for microsomal lipid peroxidation.     

The substrate binding site of human liver cytochrome P450 2C9: an NMR study

Purified recombinant human liver cytochrome P450 2C9 was produced, from expression of the corresponding cDNA in yeast, in quantities large enough for UV-visible and 1H NMR experiments. Its interaction with several substrates (tienilic acid and two derivatives, lauric acid and diclofenac) and with a specific inhibitor, sulfaphenazole, was studied by UV-visible and 1H NMR spectroscopy. At 27 degrees C, all those substrates led to an almost complete conversion of CYP 2C9 to high-spin (S = 5/2) CYP 2C9-substrate complexes characterized by a Soret peak at 390 nm; their KD values varied between 1 and 42 microM. On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon binding of its NH2 group to CYP 2C9 iron. Interactions of the five substrates with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the 1H NMR spectrum of each substrate. Distances between the heme iron atom and substrate protons were calculated from the NMR data, and the orientation of the substrate relative to iron was determined from those distances. Finally, a model for substrate positioning in the CYP 2C9 active site was constructed by molecular modeling studies under the constraint of the iron-proton distances. It points out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to establish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hydroxylation site and the anionic site. Sulfaphenazole was easily included in that model; its very high affinity for CYP 2C9 is due to a third structural feature, the presence of its NH2 function which binds to CYP 2C9 iron.     

Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes: in relation to covalent binding of its reactive intermediate

Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4-hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [3H]IMI in the presence of NADPH resulted in covalent binding of a 3H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis-Menten kinetics, and the K(m) value (1.1 microM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [3H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark Agouti < Wistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2-hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [3H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [3H]IMI metabolites covalently bound were immunoprecipitated with the anti-CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2-hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.     

Immunohistochemical localization of cytochrome P450 1A1 in precision-cut rat liver slices after in vitro exposure to beta-naphthoflavone

Mono- and polyclonal antibodies have been used to study the localization and distribution of cytochrome P450 1A1 (CYP1A1) in cultured precision-cut liver slices with various immunohistochemical methods. Neither in non-incubated slices nor in slices incubated in the absence of beta-naphthoflavone (BNF) for 24 hrs was CYP1A1 immunohistochemically detectable. After incubation in the presence of BNF (25 microM), however, CYP1A1 was well visible in parenchymal and biliary epithelial cells. CYP1A1 was not evenly distributed, but was localized predominantly in hepatocyte layers near the surfaces of the slices. This distribution could be due to the preferential uptake of BNF by outer cell layers or due to functional changes of inner cells. Together with results obtained with other methods (e.g. RT-PCR) this investigation also demonstrates that precision-cut liver slices are a useful tool for the detection of in vitro induction of CYP1A1.     

Characterization of [3H]clozapine binding sites in rat brain

We examined the characteristics of [3H]clozapine binding sites in four rat brain regions (frontal cortex, limbic area, hippocampus and striatum) in order to elucidate the pharmacological profile of this unique atypical antipsychotic drug. The specific [3H]clozapine binding was found to be saturable and reversible in all these brain regions. Scatchard analysis of the saturation data indicated that the specific binding consisted of high- and low-affinity components. Displacement experiments showed that the muscarinic cholinergic receptor represented about 50% of [3H]clozapine binding in each brain area. Serotonin 5-HT2 and dopamine D4 receptor binding sites could also be detected by displacement experiments using ketanserin and nemonapride, respectively, in frontal cortex and limbic area, but not in hippocampus or striatum. Alpha-1, alpha-2, histamine H1, dopamine D1, D2, or D3 receptor components could not be determined within the high-affinity [3H]clozapine binding sites in any brain region. It is possible that the atypical property of clozapine may depend on the modulatory effect on dopaminergic function via 5-HT2 receptor blockade and/or may be mediated via D4 receptor blockade in the mesocortical and mesolimbic area.     

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes

Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.     

Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells

Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.     

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

IBD results from the interaction of genetic and environmental factors (e.g., smoking). Clinical suspicion is the key to diagnosis, which then rests on colonoscopy, histopathological examination of multiple biopsy specimens, small bowel barium radiology and faecal examination. The primary goal of treatment is remission--histological in ulcerative colitis and symptomatic in Crohn's disease. Treating active disease and maintaining remission require different approaches. For active disease, short term corticosteroids are the mainstay of treatment, while immunosuppressive drugs are important in chronically active disease. For maintenance, mesalazine-delivering drugs and immunosuppressive agents are efficacious in both ulcerative colitis and Crohn's disease; patients with Crohn's disease should not smoke.     

Genotype of cytochrome P450 2E1 and alcohol-related blood pressure elevation in Japanese men

OBJECT: To define the longitudinal relationship of declining renal function to protein consumption and turnover in the failing renal allograft model of chronic renal failure. METHOD: The study group is our first eight consecutive cadaveric renal graft recipients who after attaining a normal creatinine clearance, then developed chronic renal failure. We analysed their urea and creatinine clearances (Cur, Ccr), serum urea (SU), urinary urea and creatinine (Ur, Ucr), serum albumin (SA), urinary protein (Upr), body weight (BW), and steroid dose. Steady state Uur is also dietary protein intake (DPI) and protein catabolic rate (PCR). Ucr measures body protein mass. Ucr/Uur measures the ratio of body protein mass to urea excretion. Mean follow-up 4.7 years, range 1.5-8.7 years. RESULTS: Mean changes: (1) Body weight (BW) rose from 56 to 65 and then fell to 61 kgms. (2) Cur fell 65 to 5 and Ccr 92 to 12 ml/min/70 kg. (3) Uur fell from 369 to 107 and Ucr from 16.8 to 9.5 mmols/day/70 kg. (4) Uur/Ucr indexed at 1:1 fell to 0.49. (5) SU rose from 8.8 to 34.9 mmol/1; SA fell from 36.1 to 31.0 gms/1; Upr rose from 1.4 to 2.3 gms/day. (6) Prednisone rose from 26 to 66 and then fell to 33 mgms/day. Correlations: (1) Cur and Uur(r = 0.99, p < 0.001). (2) Ccr and Uur (r = 0.99, p < 0.001). (3) Cur and Uur/Ucr (r = 0.88, p < 0.01) with a decelerating breakpoint at Cur 18 and Ccr 32 ml/min/70 kg (p < 0.01). (4) SU and Uur negatively (r = 0.90, p < 0.01. (5) Cur and SA albumin (r = 0.82, p < 0.05). (6) Cur and prednisone, Upr and SA do not correlate. CONCLUSIONS: In this model of chronic renal failure: (1) Renal function controls protein intake. (2) Body protein mass is relatively well preserved despite the decreased protein intake implying a decrease in the protein turnover rate and a consequent increase in body protein average age. (3) Protein malnutrition, protein ageing, and decreased protein turnover are likely pathophysiological reactions to chronic renal failure and may be part of the pathogenesis of chronic uremia.