Normal Dietary Conditions Research Articles

Salt intake and depletion increase circulating levels of endogenous ouabain in normal men

High-salt diets elevate circulating Na+ pump inhibitors, vascular resistance, and blood pressure. Ouabain induces a form of hypertension mediated via the alpha2-Na+ pump isoform and the calcium influx mode of the vascular sodium calcium exchanger (NCX). Whereas elevated levels of an endogenous ouabain (EO) and NCX have been implicated in salt-sensitive hypertension, acute changes in sodium balance do not affect plasma EO. This study investigated the impact of longer-term alterations in sodium balance on the circulating levels and renal clearance of EO in normal humans. Thirteen normal men consumed a normal diet, high-salt diet, and hydrochlorothiazide (HCTZ), each for 5-day periods to alter sodium balance. EO and other humoral and urinary variables were determined daily. On a normal diet, urinary sodium excretion (140 +/- 16 meq/day), plasma EO (0.43 +/- 0.08 nmol/l) and urinary EO excretion (1.04 +/- 0.13 nmol/day) were at steady state. On the 3rd day of a high-salt diet, urine sodium excretion (315 +/- 28 meq/day), plasma EO (5.8 +/- 2.2 nmol/l), and the urinary EO excretion (1.69 +/- 0.27 nmol/day) were significantly increased, while plasma renin activity and aldosterone levels were suppressed. The salt-evoked increase in plasma EO was greater in older individuals, in subjects whose baseline circulating EO was higher, and in those with low renal clearance. During HCTZ, body weight decreased and plasma renin activity, aldosterone, and EO (1.71 +/- 0.77 nmol/l) rose, while urinary EO excretion remained within the normal range (1.44 +/- 0.31 nmol/day). Blood pressure fell in one subject during HCTZ. HPLC of the plasma extracts showed one primary peak of EO immunoreactivity with a retention time equivalent to ouabain. High-salt diets and HCTZ raise plasma EO by stimulating EO secretion, and a J-shaped curve relates sodium balance and EO in healthy men. Under normal dietary conditions, approximately 98% of the filtered load of EO is reabsorbed by the kidney, and differences in the circulating levels of EO are strongly influenced by secretion and urinary excretion of EO. The dramatic impact of high-salt diets on plasma EO is consistent with its proposed role as a humoral vasoconstrictor that links salt intake with vascular function in hypertension.

Deficiency of Glutathione Transferase Zeta Causes Oxidative Stress and Activation of Antioxidant Response Pathways

Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity.

MECHANISMS OF DIGESTION AND ABSORPTION OF DIETARY VITAMIN A

Mechanisms involved in the digestion and absorption of dietary vitamin A require the participation of several proteins. Dietary retinyl esters are hydrolyzed in the intestine by the pancreatic enzyme, pancreatic triglyceride lipase, and intestinal brush border enzyme, phospholipase B. Unesterified retinol taken up by the enterocyte is complexed with cellular retinol-binding protein type 2 and the complex serves as a substrate for reesterification of the retinol by the enzyme lecithin:retinol acyltransferase (LRAT). The retinyl esters are then incorporated into chylomicrons, intestinal lipoproteins containing other dietary lipids, such as triglycerides, phospholipids, and free and esterified cholesterol, and apolipoprotein B. Chylomicrons containing newly absorbed retinyl esters are then secreted into the lymph. Although under normal dietary conditions much of the dietary vitamin A is absorbed via the chylomicron/lymphatic route, it is also clear that under some circumstances there is substantial absorption of unesterified retinol via the portal route. Evidence supports the idea that the cellular uptake and efflux of unesterified retinol by enterocytes is mediated by lipid transporters, but the exact number, identity, and role of these proteins is not known and is an active area of research.

A retinoid X receptor antagonist, HX531, improves leptin resistance without increasing plasma leptin level in KK-A y mice under normal dietary conditions

4-(5 H-2,3-(2,5-Dimethyl-2,5-hexano)-5-methyl-8-nitrodibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX531) is a novel retinoid X receptor antagonist. This study provides evidence that HX531 improves leptin resistance without increasing plasma leptin levels in KK-A y mice, an animal model with high plasma leptin levels and leptin resistance. Under normal dietary conditions, 3 weeks of treatment with HX531 (0.03% and 0.06% food admixture) in KK-A y mice decreased plasma leptin levels in a dose- and time-dependent manner, in addition to decreasing body weight and mesenteric fatty tissue weight. To evaluate the effect of HX531 on leptin resistance, leptin was injected intraperitoneally in the KK-A y mice for 4 days after 1 week of treatment with HX531 (0.06% food admixture). This pretreatment with HX531 resulted in exogenously administered leptin causing a significant decrease in food intake. These results suggested that HX531 decreased plasma leptin levels accompanied by a decrease in fatty tissue content in the KK-A y mice and a simultaneous improvement in leptin resistance. This is the first report that HX531 improves leptin resistance without increasing plasma leptin level in KK-A y mice, under normal dietary conditions.

Calorie restriction to retard aging and increase longevity

Calorie restriction increases longevity in rodents, delays the onset of certain diseases and has positive effects on ageing. Studies are ongoing in non-human primates. Eight persons following such a diet for 2 years reacted as anticipated (loss of body weight and fat, decrease in glycaemia and body temperature, etc.). Calorie restriction perhaps teaches us more on the means of resisting malnutrition than on ageing in normal dietary conditions. Calorie restriction is a tool for research, but it should not be recommended by practitioners, notably in elderly patients.

Inhibitor strategies to control coleopteran pests

Enzyme inhibitors from plants are promising candidates for new biocontrol agents but are limited by the inherent compensatory responses to plant inhibitors by insects. We found that inhibitors targeting multiple proteinase classes were necessary to reduce cereal damage by the red flour beetle, Tribolium castaneum. We proposed that the multiple inhibitor approach was required for efficacy because T. castaneum larvae were able to compensate when fed individual proteinase inhibitors. To investigate the adaptive response, increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to T. castaneum larvae, and the properties of digestive proteinases were examined in vitro. Under normal dietary conditions, T. castaneum larvae primarily used cysteine proteinases to digest food, with minor contributions from serine proteinases. When larvae were fed cysteine proteinase inhibitors, they responded with a dramatic shift from cysteine to serine proteinase-based digestion. A combination of cysteine and serine proteinase inhibitors in the diet prevented such a shift of dietary enzymes, resulting in a significant reduction in growth and increased mortality. These data suggest that by studying the complex of digestive proteinases in a target pest, combinations of proteinase inhibitors can be identified that overcome the adaptive response to inhibitors, thus providing a more effective control of insect pests by inhibitors.

Aluminum speciation studies in biological fluids: Part 9. A quantitative investigation of aluminum(III)–glutamate complex equilibria and their potential implications for aluminum metabolism and toxicity

As a nonessential element, aluminum is likely to be toxic both at low usual dietary levels in the long run (chronic toxicity) and at high therapeutic levels in shorter periods of time (acute toxicity). In both situations, aluminum toxicity is a direct function of aluminum bioavailability, which is itself dependent on Al 3+ solubility and charge neutralization. Dietary acids, by their intrinsic acidity and coordinating capacity, can extend the pH range, thus the section of the gastrointestinal tract, within which the Al 3+ ion remains soluble, and also help Al 3+ diffusion across the intestinal epithelium through the formation of neutral complex species. The present work examines the impact of glutamic acid, an essential amino acid also widely used in industrial food and drinks, on aluminum speciation in the gastrointestinal tract and blood plasma. Complex formation between the Al 3+ ion and glutamate has first been investigated through potentiometric titrations, complex stoichiometries being then checked by ESI mass spectrometry and NMR measurements. A series of mono- and polynuclear species has been characterized, whose influence on aluminum distribution in vivo has been assessed by computer simulation. The capacity of glutamate to maintain Al 3+ ions in solution under normal dietary conditions is predicted to be intermediate between glycine-like amino acids and succinate on the one hand, and tartrate and malate on the other hand, its Al 3+ neutralization effect being similar to that of succinate, tartrate and malate. These results, which point to a potential aggravating role of glutamate on aluminum gastrointestinal absorption, substantiate recent observations made on rats. In spite of the moderate effect expected from glutamate on aluminum bioavailability under most aluminum-based therapies investigated, attention is therefore called to the risk of glutamic acid ingestion simultaneously to any aluminum therapeutic form. Incidentally, the former implication of ‘ the’ aluminum glutamate complex in the transfer of aluminum through the blood–brain barrier of aluminum loaded rats may effectively be attributed to one of the species characterized here, but is of no significance at all to aluminum contamination in humans, even at most extreme levels.

Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells.

Previous studies indicate that the colonic H-K-ATPase mRNA is expressed as the distal nephron. However, the exact intrarenal localization of the colonic H-K-ATPase protein is still unclear. The goal of the present study was to determine the cellular and subcellular localization of the colonic H-K-ATPase protein in the rabbit kidney. We used three monoclonal antibodies (MAbs) directed against different epitopes of the rabbit colonic H-K-ATPase alpha-subunit (HKalpha(2)) to localize HKalpha(2) protein by immunofluorescence labeling of kidney sections and laser-scanning confocal microscopy. The specificity of the MAbs was confirmed by reaction with a single ~100-kDa band on Western blots of distal colon. Specific immunohistochemical reaction with the apical membrane of surface epithelial cells was observed with all three MAbs on distal colon sections. In rabbit kidney, immunofluorescence was detected only on the apical membrane of connecting tubule cells. Immunofluorescence was not detected in the cortical-, outer-, and inner-medullary collecting ducts. Furthermore, costaining with principal- and intercalated cell-specific MAbs and a MAb against the thick ascending limb suggests that these cell types express HKalpha(2) protein at levels that are below the detection limit with this method. We conclude that in the rabbit kidney, under normal dietary conditions, the HKalpha(2) protein is expressed in the apical membrane of connecting tubule cells.

Aluminum speciation studies in biological fluids. Part 7. A quantitative investigation of aluminum(III)-malate complex equilibria and their potential implications for aluminum metabolism and toxicity.

As a nonessential element, aluminum may be toxic at both environmental and therapeutic levels, depending on ligand interactions. Dietary acids that normally occur in fruits and vegetables and commonly serve as taste enhancers are good ligands of the Al(3+) ion. Malic acid is one of these and also one of the most predominant in food and beverages. The present paper reports an examination of its potential influence on aluminum bioavailability through speciation calculations based on Al(III)-malate complex formation constants especially determined for physiological conditions. According to the results obtained, malate appears to be extremely effective in maintaining Al(OH)(3) soluble over the whole pH range of the small intestine under normal dietary conditions. In addition, two neutral Al(III)--malate complexes are formed whose percentages are maximum from very low malate levels. When aluminum is administered therapeutically as its trihydroxide, the amount of metal neutralized by malate peaks as its solubility pH range regresses to its original limits in the absence of malate. The enhancing effect of malate towards aluminum absorption is therefore virtually independent of the aluminum level in the gastrointestinal tract. The presence of phosphate in the gastrointestinal juice is expected to limit the potential influence of malate on aluminum absorption. Under normal dietary conditions, phosphate effectively reduces the fraction of aluminum neutralized by malate but without nullifying it. Aluminum phosphate is predicted to precipitate when aluminum levels are raised as with the administration of aluminum hydroxide, but a significant amount of neutral aluminum malate still remains in solution. Even therapeutic aluminum phosphate is not totally safe in the presence of malate, even at low malate concentrations. As plasma simulations predict that no compensatory effect in favor of aluminum excretion may be expected from malate, simultaneous ingestion of malic acid with any therapeutic aluminum salt should preferably be avoided.

Cross-over study of the influence of bicarbonate-rich mineral water on urinary composition in comparison with sodium potassium citrate in healthy male subjects

Urine volume is the greatest risk factor for nephrolithiasis. High fluid intake is the first general advice given to stone-forming patients for the prevention of their recurrence. The aim of the present study was to evaluate the influence of bicarbonate-rich mineral water (1715 mg bicarbonate/l) on urinary-stone risk factors in comparison with sodium potassium citrate, a well-established treatment for urinary stones. The mineral water and sodium potassium citrate were administered in equimolar concentrations, with respect to the alkali load. All investigations were carried out in healthy male subjects aged 23-38 years. The study followed a cross-over design. All subjects received a standardized diet during the cross-over phase, which was formulated according to the dietary recommendations of the German Society of Nutrition (Deutsche Gesellschaft für Ernährung, 1996). On the loading day of the cross-over phase, fruit tea was substituted for either mineral water or sodium potassium citrate dissolved in fruit tea. The treatment offered during the second part of the cross-over phase was continued for a 4-week follow-up under normal dietary conditions. During the cross-over phase, there was a significant increase in urinary pH (p < 0.001). There was also a significant increase in the excretion of citric acid (P < 0.01), a decrease in the excretion of oxalic acid, and therefore a decrease in the relative supersaturations for calcium oxalate and uric acid. In the follow-up phase also, the relative supersaturations decreased and there were beneficial effects on the other urinary variables. The effect of the bicarbonate-rich mineral water was similar to that of the sodium potassium citrate, which suggests that it could be useful in the prevention of the recurrence of calcium oxalate and uric acid stones.

Absorption of Aluminium-26 in Alzheimer’s Disease, Measured Using Accelerator Mass Spectrometry

Although chromosomal abnormalities underpin some early onset cases of familial Alzheimer’s disease (AD), most cases are sporadic and not associated with such abnormalities. Aluminium (Al) is a significant but controversial risk factor for sporadic AD, and studies have reported associations between Al and the principal pathological features of AD, senile plaques and neurofibrillary tangles. The present study measured gastrointestinal (GI) absorption of Al under normal dietary conditions using ²⁶Al tracer and accelerator mass spectrometry (AMS). Following overnight fast, 13 AD patients (aged 63–76 years) and 13 age-matched controls (aged 62–76 years) ingested a fruit drink containing 27 ng ²⁶Al. Plasma samples were obtained before and 1 h after the drink and from these the fraction of ²⁶Al absorbed across the GI tract was estimated. The GI tract rigorously excludes Al with only 0.06–0.1% of the ingested Al being absorbed. The mean fraction absorbed by AD subjects exceeded controls by a factor of 1.64 (p≤0.05, Anova). AMS is capable of determining <10^–16 g of ²⁶Al with many orders of magnitude more sensitivity than other techniques. Using this sensitivity, we have shown, under normal physiological conditions, that the ability of the GI tract to exclude Al is reduced in AD, possibly leading to greater systemic exposure to Al. Public health measures to limit Al dietary uptake or bioavailability may decrease the prevalence of AD in the community and should be considered.

The major low molecular weight apolipoprotein from normal and hyperlipidemia atherosclerosis-prone (LAP) Japanese quail

The low molecular weight (LMW) apolipoprotein of apo C plays an important role in the metabolism of triglyceride-rich lipoproteins. This study aimed at a characterization of the major LMW apolipoproteins from normal quail strain, and also from LAP (hyperlipidemia atherosclerosis-prone) strain to identify its genetic disorder. The major LMW apoprotein cDNA clone from normal quail comprised of approximately 500 bp, and encoded polypeptide of 78 amino acid residues containing 57 amino acids as a mature apolipoprotein. Although the quail LMW apoprotein showed a low homology to either apo C-I, C-II, or C-III of other animals, it retained a well-developed amphipathic α-helix structure. There was no difference in the deduced primary structure of the quail LMW apoprotein between LAP and normal strain. An analysis of the mRNA expression showed that the quail LMW apoprotein was only expressed in the liver of both LAP and normal Japanese quail. No difference was noted in the hepatic expression of the quail LMW apoprotein mRNA between normal and LAP strains with neither normal nor atherogenic dietary conditions. The structure and expression of the major LMW apoprotein thus had no relevance to higher susceptibility of LAP strain to the experimental atherosclerosis.

A CASE OF PICA AND IRON DEFICIENCY ANAEMIA IN NOTTINGHAM

SUMMARYPica is the persistent eating of a non‐nutritional substance (as defined by the American Psychiatric Association) and is often associated with iron deficiency. Current literature leaves uncertain whether pica causes iron deficiency via its proposed effect on iron absorption or, conversely, whether iron deficiency causes pica. We report the case of a young girl from Nottingham who we believe developed pica secondary to her iron deficiency anaemia. To determine that her pica was not affecting gastrointestinal iron absorption we carried out iron absorption studies first under pica and then under normal dietary conditions.

Effects of dietary manipulations and glucose infusion on glucagon response during exercise in rats.

The purpose of the present investigation was to test the hypothesis that blood glucose concentration is not always related to glucagon response during exercise. Three groups of rats were submitted to a prolonged (3-h) swimming exercise. Two groups of rats had their normal food intake restricted by 50% the night before the experiment. One of these two groups of rats was intravenously infused with glucose throughout exercise to maintain euglycemia. The third group of rats swam while under normal dietary conditions. Plasma glucose, sampled in arterial blood, was reduced (P < 0.05) at 75, 105, 150, and 170 min of exercise (from approximately 130 to 110 mg/dl) in the food-restricted animals without glucose infusion, whereas a significant (P < 0.05) increase was measured in the two other groups during exercise. A significant (P < 0.01) difference in the mean integrated areas under the glucose-concentration curve was found only between the fed and the two food-restricted groups. Plasma insulin concentrations decreased (P < 0.05) similarly in all groups during exercise, whereas plasma epinephrine and norepinephrine concentrations increased significantly (P < 0.01) in all groups. Despite differences between groups in plasma glucose response during exercise, and despite the absence of any decrease in exercising blood glucose levels in at least two of the three groups, plasma glucagon responses were increased (P < 0.05) similarly in all groups (from approximately 250 to 550 pg/ml) at the end of the exercise period. The increase in glucagon was significant after 90 min of exercise in the food-restricted groups, with or without glucose infusion, but only after 140 min in the fed group. These results indicate that the glucagon response during exercise is not always linked to the decrease in plasma glucose.

Gastrointestinal absorption of aluminum is increased in down's syndrome

Individuals with Down's Syndrome (DS) develop the neuropathological features of senile dementia of the Alzheimer's type (SDAT) by early middle age. Because of recent evidence that gastrointestinal (GI) aluminum (Al) absorption is increased in patients with SDAT, and that Al may contribute to associated neuropathological changes, we have investigated the GI uptake of Al in patients with DS by two methods. The first measured the absorption of 27Al at concentrations associated with antacid use, in the presence of citrate, using atomic absorption spectrometry. There was no difference between basal blood concentrations of 27Al in 15 DS subjects (36-46 years) and 15 age-matched controls. The mean increase in 27Al blood concentrations 60 minutes after the dose of Al was four times greater in the DS group than in controls (p < 0.001). The second measured GI absorption of 26Al under normal dietary conditions using accelerator mass spectrometry. With 26Al the mean Al absorption in DS subjects (n = 5) exceeded that of controls (n = 4) by a factor of 6 (p < 0.02). Although the mechanisms of enhanced absorption are unknown, the data indicate that similar abnormalities in the GI handling of Al occur in both SDAT and DS suggesting that it may be advisable to minimize dietary exposure to Al in subjects at risk of developing Alzheimer-type pathology.

The effects of N-methyl-d,l-aspartic acid and aspartic acid on the plasma concentration of gonadotrophins, GH and prolactin in the ewe

Aspartic acid is a neurotransmitter in the central nervous system that acts via the glutamate receptor and the analogue, N-methyl-D,L-aspartic acid (NMA) is an agonist that stimulates GnRH secretion. Under normal dietary conditions, the plasma concentration of aspartic acid in ewes is low and if increased by improved nutrition may increase the brain concentration of aspartic acid leading to increased gonadotrophin secretion. In two experiments we investigated the effects of NMA on pituitary hormone concentrations and the effects of aspartic acid on ovulation rate and pituitary hormone concentrations. The intravenous injection of NMA into cycling ewes resulted in an immediate (within 15 min) release of a pulse of LH and of GH and a prolonged (up to 1 h) suppression of prolactin secretion. There were marked differences in responsiveness to NMA between individual ewes. The intravenous infusion of aspartic acid for 5 days in the late luteal phase of the oestrous cycle did not affect ovulation rate but reduced the mean LH (P < 0.05) and FSH (P < 0.05) concentrations in plasma. The frequency of LH pulses also tended to be lower (P < 0.1) in ewes infused with aspartic acid. It is suggested that the decrease in gonadotrophin secretion in ewes infused with aspartic acid is due to effects on the hypothalamus or the anterior pituitary gland which are not related to increased levels of ovarian feedback. These changes are likely to involve decreased GnRH secretion.

Increase in retinyl palmitate concentration in eyes and livers and the concentration of interphotoreceptor retinoid-binding protein in eyes of vitiligo mutant mice.

Retinyl esters play an important role in the visual cycle because they are involved in regeneration of 11-cis-retinal for use in rhodopsin formation. In the present study, retinyl ester concentrations were significantly elevated in eyes and livers of mice homozygous for the vitiligo mutation (mivit/mivit). Vitiligo mice demonstrate a slowly progressing retinal degeneration characterized by gradual loss of photoreceptor cells and rhodopsin as well as uneven pigmentation of the retinal pigment epithelium (RPE). Analysis of retinoids by h.p.l.c. indicated that the retinyl palmitate level was increased fivefold in eyes of affected mice at 10 weeks postnatally and was threefold higher at 22 weeks of age. Accumulation of retinyl palmitate occurred in the RPE rather than the neural retina. Furthermore, the concentration of all-trans-retinol was elevated in the RPE of vitiligo mice. Levels of interphotoreceptor retinoid binding protein (IRBP) were increased in vitiligo mice between ages 4 and 14 weeks, but returned to normal by 16 weeks. Increased IRBP levels were not due to increased protein synthesis because IRBP mRNA levels did not differ significantly between control and affected animals. To examine possible systemic involvement in vitiligo mice, retinoids were evaluated in liver and plasma. Mean hepatic total vitamin A levels in affected mice were approximately 1.7 times higher than controls. Analysis of esterified and non-esterified retinoids in liver showed that the concentration of retinyl palmitate was elevated. Plasma retinol levels were normal. This study provides the first evidence of altered systemic retinoid metabolism in vitiligo mice, which occurs, significantly, under normal dietary conditions.

The absorption of phosphate from the digestive tract of ruminant animals

Under normal dietary conditions in which the phosphorus (P) intake of ruminant animals is adequate, the absorption of phosphate (Pi) ions takes place from both the fore-stomachs and the small intestine. The rate of Pi absorption from the latter appears to be at least three times greater than that from the former. When absorption of Pi ions from the reticulorumen is severely reduced, as in dietary P deficiency, the absorption of both calcium and magnesium ions from the reticulorumen becomes impaired. Thus, dietary P deficiency may play a role in the aetiology of acute grass tetany.

Metabolic Regulation and Transgenic Mouse.

The ornithine-urea cycle is the major pathway for detoxication of ammonia formed in amino acid metabolism and is responsible for nitrogen balance of the body. The cycle is also involved in biosynthesis of arginine. Ornithine transcarbamylase (OTC) catalyzes the second among the five enzymatic steps of the cycle. The enzyme is present in the liver and to a lesser extent in the small intestine. In the fetal liver, the level of the enzyme increases late in gestation, coordinately with other urea cycle enzymes. OTC deficiency is known in humans and mice, as an inborn error of metabolism that results in protein intolerance and hyperammonemia. The rat and human OTC genes span a region of 70-75 kb on the X chromosome. Transient expression analysis showed that the promoter of this gene is much more active in a hepatoma cell line (HepG2) than in a non-hepatic cell line (CHO) . Two sites of the promoter region are recognized by both HNF-4 (hepa-tocyte nuclear factor-4) and COUP-TF (chicken ovalbumin upstream promoter-transcription factor) that are members of steroid receptor superfamily. In cotransfection analysis, HNF-4 activated expression from the OTC promoter, whereas COUP-TF repressed it. In transgenic mice, the promoter of the rat OTC gene directed liver-and small intestine-specific expression, but the expression of the transgene in the liver was very low. We found a hepatoma-specific enhancer region of -110 by located 11 kb upstream of the transcription start site. There are four protein binding sites in this region; two were recognized by HNF-4 and the other two by a factor (s) related to C/EBP (CCAAT/enhancer binding protein) . Both HNF-4 and C/EBP sites were required for the enhancer activity. In transgenic mice, an OTC enhancer/OTC promoter/OTC cDNA transgene was expressed more strongly in the liver than in the small intestine, thus resembling the expression of the endogenous gene. Introduction of the OTC promoter/OTC cDNA transgene into the OTC-deficient spf-ash mouse apparently corrected the OTC deficiency under normal dietary conditions. However, this transgenic spf-ash mouse was not completely healthy under stressed conditions. Introduction of the OTC enhancer-containing transgene into the OTC deficiency mouse is underway.

The fish odour syndrome: biochemical, familial, and clinical aspects.

To study the biochemical, familial, and clinical features of the fish odour syndrome among subjects with suspected body malodour. Subjects who responded to a newspaper article were screened for the fish odour syndrome by interview and biochemical tests. Families of subjects with the syndrome were tested if possible. St Mary's Hospital, London, and some interviews at subjects' homes. 187 subjects (28 males) with suspected body malodour, of whom 156 (19 males) underwent biochemical tests. Five families of six of the subjects with the fish odour syndrome agreed to further tests. Amounts of trimethylamine and trimethylamine N-oxide in urine collected over 24 hours under normal dietary conditions and for eight hours after oral challenge with 600 mg trimethylamine. The fish odour syndrome was diagnosed in 11 subjects: the percentage of total trimethylamine excreted in their urine samples that was oxidised to trimethylamine N-oxide was < 55% under normal dietary conditions and < 25% after oral challenge with trimethylamine (in normal subjects > 80% of trimethylamine was N-oxidised). Parents of six of the subjects with the syndrome were tested: all showed impaired N-oxidation of excreted trimethylamine (< 80%) after oral challenge, indicating that they were heterozygous carriers of the allele for the syndrome. The syndrome was associated with various psychosocial reactions including clinical depression. The fish odour syndrome can be inherited in an autosomal recessive fashion. It should be considered as a possible causative factor in patients complaining of body malodour.