Maurice J. Arnaud

Assessment of the cytochrome P-448 dependent liver enzyme system by a caffeine breath test.

Summary[1-Methyl-14C], [3-Methyl-14C] and [7-Methyl-14C] caffeine were used to investigate demethylation in control rats, and in rats pretreated with phenobarbital or 3-methylcholanthrene, by a14CO2-exhalation test. Compared to controls, pretreatment with phenobarbital did not enhance demethylation of any of the labelled caffeines. In contrast, induction by 3-methylcholanthrene, presumably of cytochrome P-448, resulted in highly significant increases in peak14CO2 exhalation rates,14CO2 disappearance constants and areas under the exhalation rate — time curves. Based on these results, [7-methyl-14C] and [3-methyl-14C] caffeine were chosen for assessing the feasibility of a caffeine breath test in man, using 5 normal volunteers and 2 patients with compensated liver cirrhosis.14CO2 exhalation curves in cirrhotics were clearly different from those in normal volunteers, being characterised by a slower rise and a lower specific activity of exhaled14CO2. Since the variability of the levels of the specific activity in subjects with normal livers suggested the influence of extraneous factors, a second group of normal volunteers, smokers and nonsmokers, was investigated. With either labels, the average14CO2 exhalation rate was doubled in smokers. From these studies in rats and preliminary results in man it is concluded that specifically labelled caffeine is a suitable and promising substrate for studying demethylation by breath analysis. Presumably, caffeine represents a safe and sensitive indicator of the activity of the cytochrome P-448 system.

Identification, kinetic and quantitative study of [2-14C] and [1-Me-14C]caffeine metabolites in rat's urine by chromatographic separations

The urinary metabolites of [2-14C] and [1-Me-14C]caffeine administered orally have been separated by two-dimensional thin-layer chromatography from whole urine, and localized by autoradiography. The scanning of chromatograms gives quantitative results showing the importance of the metabolic pathway leading to trimethyldihydrouric acid. The following three new metabolites were identified in rats urine: 1,7-dimethyluric, N-methylurea, and NN′-dimethylurea. The excretion kinetics of each metabolite show that caffeine, theophylline, trimethylallantoin, and an unknown derivative which is either an isomer or a precursor of trimethylallantoin were excreted later than the other metabolites as a whole.

Mineral water intake reduces blood pressure among subjects with low urinary magnesium and calcium levels

BackgroundSeveral previous epidemiological studies have shown a relation between drinking water quality and death in cardiovascular disease whereas others have not found such a relationship. An intervention study was undertaken to evaluate the effect of water with added magnesium and natural mineral water on blood pressure.MethodsA group of 70 subjects with borderline hypertension was recruited and consumed 1) a water low in minerals, 2) magnesium enriched water or 3) natural mineral water, in a random, double blind fashion during four weeks.ResultsAmong persons with an initial low excretion of magnesium or calcium in the urine, the urinary excretion of magnesium was increased in the groups consuming the two waters containing magnesium after 4 weeks. A significant decrease in blood pressure was found in the group consuming mineral water at 2 and 4 weeks.ConclusionThe results suggest that minerals taken in water are significant for the body burden and that an intake of mineral water among persons with a low urinary excretion of magnesium or calcium may decrease the blood pressure. Further studies should investigate the extent of mineral deficiency in different populations and the efficiency of different vehicles for supplying minerals, particularly magnesium and calcium.

Rapid and complete urinary elimination of [14C]‐5‐hydroxymethyl‐2‐furaldehyde administered orally or intravenously to rats

5-Hydroxymethyl-2-furaldehyde (HMF), is a major product of sugar degradation found in food and solutions used in parenteral nutrition. Labeled [14C]HMF was synthesized by dehydration of [14C]fructose on ion-exchange resin and administered per os (po) and intravenously (iv) to rats. Metabolic balance of radioactivity demonstrated that HMF or its metabolites are rapidly eliminated in the urine with a recovery of 95-100% after 24 h. Literature reported, in some cases, 50% retention in the body. HMF was completely converted to two metabolites, which have been identified by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) as 5-hydroxymethyl-2-furoic acid and N-(5-hydroxymethyl-2-furoyl)glycine. Administration of high doses of HMF showed a similar rapid elimination, but a proportional reduction of the amount of the glycine conjugate produced. Whole-animal-body autoradiography confirm that shortly after administration radioactive material was present in the liver but was mostly in the kidney and the bladder. The only significant difference between po and iv administration was the presence of a higher level of radioactive material in the brain of iv-treated rats.

High levels of methylxanthines in chocolate do not alter theobromine disposition

Theobromine disposition was measured twice in 12 normal men, once after 14 days of abstention from all methylxanthines and once after 1 week of theobromine (6 mg/kg/day) in the form of dark chocolate. Mean theobromine t½, apparent volume of distribution, and clearance after abstinence from all methylxanthines were 10.0 hours, 0.76 L/kg, and 0.88 ml/min/kg. High daily doses of chocolate for 1 week did not change these values. After subjects abstained from methylxanthines, urinary radioactivity over 72 hours after a single, oral dose of [8‐14C]theobromine consisted of 42% 7‐methylxanthine, 20% 3‐methylxanthine, 18% theobromine, 10% 7‐methyluric acid, and 10% 6‐amino‐5[N methyl‐formylamino]‐1‐methyluracil. A week of daily theobromine consumption in the form of dark chocolate also did not alter this urinary profile of theobromine and its metabolites. Although these results might appear to differ from other reports of inhibition of theobromine elimination after five consecutive daily doses of theobromine in aqueous suspensions, both the rate and extent of absorption of theobromine in chocolate were less then that of theobromine in solution. Relative bioavailability of theobromine in chocolate was 80% that of theobromine in solution. This reinforces the fundamental principle that both the metabolic and the therapeutic consequences of a particular chemical can differ when that chemical is given in the pure compared with the dietary form.

CYP1A2 genetic polymorphisms and adenocarcinoma lung cancer risk in the Tunisian population.

AIMS In this study, the effects of four single nucleotide polymorphisms (SNPs), -3860G>A, -2467delT, -739T>G and -163C>A, of CYP1A2 gene on lung cancer were evaluated in Tunisian population. MAIN METHODS Four polymorphisms of CYP1A2 gene were analysed in 109 healthy smokers and in 101 lung cancer cases, including 63 with squamous cell carcinoma (SCC) and 41 with adenocarcinoma (AD). The genotyping for the SNPs -3860 G>A, -2467delT, -739T>G and -163C>A was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. KEY FINDINGS The results showed that smokers with CYP1A2 gene polymorphisms were associated with an increased risk for the development of lung AD. There was however no significant increased risk of developing lung SCC in smokers having CYP1A2 gene polymorphisms. An increased risk of developing AD was observed in smokers who are carriers of at least one copy of -3680A or -739G giving a significant odds ratio (OR) of 6.02 (CI=2.91-12.9) and 3.01 (CI=1.54-5.98), respectively. SIGNIFICANCE These genotyping data are consistent with the hypothesis that tobacco-specific-N-nitrosamines (TSN) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are major contributors to the development of lung AD and that CYP1A2 gene product plays an important role in the metabolic activation of NNK. This study suggests that SNPs of CYP1A2 could be considered as promising biomarkers in the aetiology of lung AD in smokers.

Placental transfer of the major caffeine metabolite in the rat using 6-amino-5[N-formylmethylamino]1,3[Me-14C]-dimethyluracil administered orally or intravenously to the pregnant rat

6-Amino-5[N-formylmethylamino]1,3[Me-14C]dimethyluracil (1,3,7-DAU), the most important caffeine metabolite in the rat and a minor one in man was synthesized and administered p.o. or i.v. to pregnant rats. This study demonstrates the distribution of this metabolite in the animal and its transfer to the embryos and the fetus. The fetus was shown to be protected by a placental barrier which leads to a lower fetal tissue exposure 1 h after the administration, the equilibrium between fetus and pregnant rat being reached 4-5 h later. Future studies testing the fetotoxicity of this metabolite compared with caffeine must take into consideration that only about half of the oral dose is absorbed. In addition, similar fetal tissue exposure must be obtained when this metabolite is given orally or is produced from caffeine.

Comparison of carbohydrate utilization in man using indirect calorimetry and mass spectrometry after an oral load of 100 g naturally-labelled [ 13 C]glucose

1. Carbohydrate (CHO) oxidation was measured simultaneously in a group of five normal subjects after an oral load of 100 g naturally-labelled [13C]glucose, using indirect calorimetry and mass spectrometry. 2. CHO utilization, calculated from the results of indirect calorimetry, increased 30 min after the glucose load to reach a peak at 90 min. It then decreased to reach basal values at 380 min. Cumulative total CHO oxidation at 480 min was 83 +/- 8 g, and CHO oxidized above basal levels, 37 +/- 3 g. 3. Enrichment of expired carbon dioxide with 13C began at 60 min and maximum values were observed at 270 min. At 480 min, cumulative CHO oxidation measured by use of [13C]glucose was 29 g. The difference from calorimetric values can be attributed in part to the slow isotopic dilution in the glucose and bicarbonate pools. 4. Thus, approximately 30% of the glucose load was oxidized during the 8 h after its ingestion and this accounts for a significant part of the increased CHO oxidation (37 g), as measured by indirect calorimetry.

Renal effects of low-dose aminophylline and enprofylline in newborn rabbits

Renal function was studied in 15 newborn New Zealand rabbits administered either 0.6 mg/kg enprofylline intravenously. Each animal acted as its own control. Glomerular filtration rate (GFR) and renal blood flow (RBF) were assessed by the clearances of inulin and para-aminohippuric acid, respectively. Enprofylline, a xanthine with low adenosine antagonistic properties, did not modify urine flow rate, GFR, RBF, renal vascular resistance, filtration fraction, sodium and potassium urinary excretion, whereas administration of theophylline, a potent adenosine antagonist, was associated with a significant increase in diuresis, renal vascular resistance and filtration fraction. The differences observed in the renal effects of theophylline and enprofylline strongly support the view that 1) the renal actions of micromolar concentrations of theophylline are mediated by an antagonism with endogenous renal adenosine; 2) renal adenosine could play a physiological role in the regulation of renal hemodynamics.

Metabolism and Distribution of Labeled Theophylline in the Pregnant Rat. Impairment of Theophylline Metabolism by Pregnancy and Absence of a Blood-Brain Barrier in the Fetus

Summary: Theophylline was shown to cross the placenta and to be distributed after 1 h among the organs of the fetus and the pregnant rat except for the brain. Lower radioactivity was found in the central nervous system of the pregnant rat compared to the other organs and particularly the brain of the fetus. The blood-brain barrier was observed for theophylline in the adult rat. The degree of exposure of the fetal brain is shown to be twice that of the adult brain. The total absence of a blood-brain barrier for the fetus is thus demonstrated. A ratio of brain to blood theophylline concentration of 0.40 and 0.87 was found respectively for the pregnant rat and the fetus. The presence of a blood-brain barrier observed in the rat cannot be extrapolated to the human. Either the absence or a less efficient blood-brain barrier in the human fetus and neonate has to be taken into consideration. After theophylline administration to pregnant asthmatic women, some of the neonates displayed clinical symptoms that may have been related to placental transfer of theophylline (7, 37).The ability of rat fetus to methylate theophylline into caffeine is demonstrated. Traces of caffeine are produced in utero that may be explained by the absence of theophylline accumulation in the fetus.Urinalysis of the metabolites showed that unchanged theophylline amounted to 73 ± 6% of total urine radioactivity in the pregnant rat as compared to 35 ± 3% in the non-pregnant animal. This impairment of theophylline metabolism in late pregnancy is explained by the decreased formation of 1,3-dimethyluric acid (−68%). The production of 1-methyluric acid was less affected (−30%). Modification of theophylline metabolism during human pregnancy has never been studied. Although 1,3-dimethyluric is the most important theophylline metabolite both in the rat and man, there are such important species differences in the quantitative metabolic pathways that it is important to show whether or not the results obtained from rat studies can be extrapolated to the human.Speculation: In the human, the exposure of the fetus to impaired theophylline metabolism during pregnancy or the absence of a blood-brain barrier could explain clinical symptoms of neurotoxia observed in the neonate when theophylline was administered to pregnant women.