Caffeine as a probe for CYP1A2 activity: potential influence of renal factors on urinary phenotypic trait measurements.

Two established caffeine-based urinary methods for measuring CYP1A2 activity were compared with each other, and also with the systemic clearance of caffeine which served as a standard of reference for such activity. Following a standardized dose, caffeine (137X) and its metabolites were measured in urine and plasma of 39 healthy subjects. The measurements allowed determinations of: (1) systemic caffeine clearance (CL(caff)); (2) the caffeine metabolite ratio (AFMU + 1X + 1U)/17U determined in an overnight-urine specimen and referred to as CMR, and (3) the ratio (17X + 17U)/137X measured in urine collected between 4 and 5 h after caffeine intake and referred to as PCUR for 'paraxanthine-caffeine urinary ratio'. The PCUR showed a bimodal distribution and a relatively wide variation, CL(caff) and CMR were both normally distributed. The correlation between CL(caff) and CMR was r = 0.77 (p < 0.001), between CLcaff and PCUR r = 0.46 (p < 0.01), and between CMR and PCUR r = 0.40 (p < 0.02). The difference between the correlation coefficients 0.77 and 0.46 was statistically significant (z-test; p < 0.05). The well established decrease of caffeine metabolism by oral contraceptive use was observed with both CL(caff) and CMR but not with PCUR. Examination of possible explanations for the differences between PCUR and CMR led to the finding of a correlation between PCUR and the renal clearance of caffeine (CLr) with r = -0.47 (p < 0.01). Further scrutiny demonstrated that a bimodal or non-normal frequency distribution as shown by PCUR was also shown by CLr and by urine flow rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonyl (phenone) reductase in human liver: inter-individual variability.

The enzyme family carbonyl reductase, which catalyses the reduction of xenobiotic as well as endogenous ketones and aldehydes, has not been very well studied in terms of its biological functions and structural aspects. The aim of the present study was to check for the occurrence of inter-individual variability of carbonyl reductase activity in human liver. In vitro metabolism of p-nitroacetophenone (PNAP, a prototype substrate) indicated the presence of a high- and low-affinity enzyme site. The reductase activity of 17 kidney donor livers was screened at two concentrations (0.05 and 0.5 mM PNAP, below and above Km). The rates of reductase activity at 0.05 mM suggested a normal distribution. In contrast, at 0.5 mM the rates indicated a non-normal distribution, i.e. bi- or tri-modality. As an index of variability of enzyme affinity, ratios of velocities at 0.5 to 0.05 mM PNAP were calculated in order to check their frequency distributions. Three out of 17 kidney donor livers showed an atypical ratio. In these three cases, the high ratio was due to the low activity of the high affinity form of carbonyl reductase. Autopsy livers are a more readily available tissue source and about half the activity of the kidney donor livers was found in 43 autopsy livers indicating that they are a useful source of human tissue for studies of carbonyl reductase.

Acute and latent leukopenic reaction to antipyrine.

During the past 10 yr over 100 subjects received test doses of antipyrine in our laboratory for drug metabolism studies without any noticeable untoward effects. The present case describes an immediate allergic reaction to antipyrine and a latent leukopenic reaction 8 wk later without any drug exposure. Leukoagglutination was demonstrated in vitro following the addition of antipyrine, aminopyrine, phenylbutazone, or sulfinpyrazone to blood taken from the subject.

An alternative test for acetylator phenotyping with caffeine.

Previously published methods allow the determination of the genetically controlled acetylator status using caffeine as a test drug, based on the urinary excretion of a ring-opened metabolite of caffeine, an acetylated uracil (5-acetylamino-6-formylamino-3-methyluracil). 5-Acetylamino-6-formylamino-3-methyluracil is labile but can be converted into a stable, deformylated product referred to as 5-acetylamino-6-amino-3-methyluracil, which has recently been shown to be quantifiable by exclusion chromatography. The first part of the present article represents a longitudinal study of three subjects to assess the intraindividual variability of those caffeine metabolite ratios that are of potential interest for the determination of acetylator phenotypes. Effects of single and multiple doses, as well as of different periods of urine collection, were tested. A ratio relating the excretion of 5-acetylamino-6-amino-3-methyluracil to that of all products of the 7-demethylation pathway of paraxanthine proved to be highly reproducible, particularly after collection of overnight urine after coffee consumption during the day. This ratio showed complete concordance with the plasma index for sulfamethazine acetylation. The second part of this article showed the use of this ratio in a population study. It allowed a good separation of slow and fast acetylators and probably also a separation of homozygous and heterozygous fast acetylators.

A case of deficiency of N-hydroxylation of amobarbital.

It has been shown recently that the overall metabolism of amobarbital in man is essentially under genetic control. The drug normally undergoes two hydroxylation reactions, leading to 3'-hydroxyamobarbital (C-OH) and N-hydroxyamobarbital (N-OH). This paper describes a sibship in which two mothers who are identical twins show a gross deficiency on N-OH elimination in urine. The whole set of sibship data suggests that this deficiency represents a recessive trait controlled by a single pair of allelic autosomal genes which regulate N-OH formation. Several methodical approaches to assess an individual's capacity for N-OH formation are illustrated. There was no evidence of compensatory or concordant regulation of the two hydroxylation reactions. The case of this family illustrates that the functional lack of a biotransformation reaction is almost certain to be overlooked if one measures only the disappearance of a multimetabolized drug and not the appearance of metabolites.

Distinctive patterns of amobarbital metabolites.

This paper establishes that the relative proportion of amobarbital metabolites in urine is highly variable from person to person and that observations of plasma half-life give no indication of this variability, but it shows that a valid estimate of a given person's metabolite pattern can be obtained by studying a single urine specimen in the postdistributive phase. The two metabolites which were measured in urine accounted on the average of 9 subjects for 80% +/- 3% of the dose with a range from 66% to 94%. The two metabolites were the well known 3'-hydroxyamobarbital (COH) as a product of side chain hydroxylation and N-beta-D-glucopyranosyl amobarbital (N-glu), a glucose conjugate which at some earlier time had been mistaken for an N-hydroxylation product. Among 129 volunteer subjects, the metabolite ratio N-glu/COH showed a median value of about 0.5 with a range from 0 to 2.8. A virtual absence of N-glu was observed in one of the 129 subjects and confirmed by a second administration of amobarbital 3 mo later. Of the 14 subjects with predominant N-glu excretion 4 were of Chinese origin, while there were 6 Chinese among the 115 other subjects (p less than 0.02).

Variation in amobarbital metabolism: evaluation of a simplified population study.

Kinetic constants of amobarbital metabolism were established for 52 subjects on the basis of urinary analysis extending over several days, usually 96 hr. There was no evidence of effect of age or sex on any of the constants. C-Hydroxylation was induced by cigarette smoking as much as 100%, but glucosidation was not affected. A factor influencing the constants was ethnicity of subjects (Caucasian or Oriental). This study confirms ethnic differences in amobarbital metabolism that were reported after measuring the concentration of metabolites in single samples of urine, that is, urine specimens voided during the postdistributive phase after oral drug intake. It appears that extreme simplification of sampling methods may be contemplated in the design of metabolic investigations of populations.

A method for studying drug metabolism in populations: racial differences in amobarbital metabolism.

The two main metabolites of amobarbital excreted in urine are 3'-hydroxyamobarbital (C-OH) and 1-(beta-D-glucopyranosyl) amobarbital (N-glu). When testing the metabolite ratio in small single samples of urine, it was found that the urine in a Caucasian population contained about one-third glucose conjugation and two-thirds hydroxylation product, while an Oriental population excreted both metabolites in equal proportion. Attempts to learn the causes for the different metabolite ratios led to an investigation of metabolite concentrations in urine. The sums of the average urinary concentration of C-OH was greater in Caucasians than in Orientals, no matter how the data were expressed; the reverse was true for the N-glu metabolite. C-OH data was scattered more widely among Orientals than Caucasians; this might indicate bimodality of the distribution curves. There also was a trend toward more N-glu metabolite in urine of females than of males. Measuring the metabolite/creatinine ratios narrowed the distribution range of the data, particularly after correction for sex difference in creatinine, but population differences were not changed. Expected relationships between metabolite content of urine, sampling times, and plasma half-life (t1/2) were established by calculation. A Caucasian female with no capacity for N-glucosidation was found during the first part of this population survey. An Oriental male with only trace capacity for amobarbital hydroxylation was found in the second part.

Inter- and intrasubject variation in diazepam free fraction.

The extent of intersubject variation in diazepam free fraction was measured in fasting plasma of 74 unrelated subjects. Free fraction differences between subjects were significant and ranged from 0.97% to 1.99%. Diazepam free fraction in 29 males was normally distributed about a mean of 1.25% (range, 1.05% to 1.47%), but the distribution in females was skewed to higher free fractions and 40% had values above the highest in males. Albumin concentration (r = -0.27, p less than 0.002) and age (r = 0.44, p less than 0.001) only accounted for a small part of the variation. Within-pair variances were not greater in 11 dizygotic than in 18 monozygotic twin pairs, indicating a greater contribution of environmental than of genetic factors to diazepam binding. The prehemodialysis free fractions of diazepam in 9 uremic patients ranged from 3.44% to 6.69%, and decreased (p less than 0.005) in 7 after 6 hr of hemodialysis. In 10 subjects determination of intrasubject variation in diazepam free fraction between 14-hr fasting and 2-hr postprandial plasma samples indicated that because subjects differ in their pattern of change in free fraction (p less than 0.001), the overall decrease in mean free fraction did not achieve statistical significance (p = 0.10). The mean relative percent change in free fraction within subjects after feeding was 15.2%.

Caffeine as a metabolic probe: validation of its use for acetylator phenotyping.

The use of two caffeine metabolite ratios for acetylator phenotyping was validated by demonstrating concordance with two sulfamethazine tests in 178 unrelated healthy subjects. The caffeine metabolites used for this purpose were 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylxanthine (1X), and 1-methylurate (1U). The ratio AAMU/(AAMU + 1X + 1U), referred to as molar ratio or N-acetyltransferase, was compared with the ratio AAMU/1X. The results indicated that, for screening purposes, the acetylator phenotype can be determined by analysis of a 6-hour urine sample after a cup of coffee or strong tea or a can of caffeine-containing soft drink. The ratio AAMU/1X is the ratio of choice for the study of subjects in whom variability of xanthine oxidase can be neglected; use of the ratio AAMU/(AAMU + 1X + 1U) appears appropriate for special purposes. Gender, ethnic origin, habitual or moderate consumption of coffee, tea, soft drinks, or ethanol, or cigarette smoking have little if any effect on the caffeine tests for acetylator phenotyping.

Genetic polymorphism of mephenytoin p(4')-hydroxylation: difference between Orientals and Caucasians.

The genetically controlled mephenytoin p(4')-hydroxylation capacity was determined in 118 Caucasians and 70 Orientals. After an oral dose of 50 or 100 mg of racemic mephenytoin, the amount of p(4')-hydroxymephenytoin in 24 h urine was measured by gas chromatography. Bimodal distribution was found with 9/70 (13%) Orientals and 5/118 (4%) Caucasians demonstrating deficient p(4')-hydroxylation. The statistically significant difference between Orientals and Caucasians (P less than 0.05) was accounted for by the high incidence of poor metabolizers among the Japanese subjects, 7/31 (23%). The frequency among Chinese subjects, 2/39 (5%), was similar to the frequency among Caucasians.

The fate of [7-3H]isoproterenol in cats after intravenous administration.

The fate of intravenously administered [7-3H]isoproterenol was investigated in cats. Ten minutes after injection the concentration of 3H was highest in the heart, lungs, adrenals, and kidneys, but after 5 hr most of the radioactivity was found in the liver. The concentration of the unchanged drug in the serum declined in a biphasic manner with half-lives of 2.1--2.5 min for the first phase, and 58--77 min for the second phase. The drug was rapidly metabolized to 3-O-methylisoproterenol (MISP) and then conjugated. In 5 hr 44--55% of the administered 3H was excreted in the urine, 2--2.5% as unchanged drug, 21--41% as MISP, and 12--22% as conjugated MISP. Conjugated MISP was also found in the bile. The results indicate that the rate of formation of MISP in cats is much faster than its rate of conjugation and excretion.

The fate of orally administered [4-14C]phenytoin in two healthy male volunteers.

A recovery study was conducted to determine whether phenytoin (DPH), like the barbiturates, is metabolized via the recently discovered N-glucosidation pathway. Virtually 100% of the ingested 14C-labelled doses in two subjects could be accounted for in the excreta within 5 days, with 35% in feces and 65% in urine. Radioactivity in the urine was entirely due to free and conjugated 5-(4-hydroxy-phenyl)-5-phenylhydantoin (p-HPPH) and the dihydrodiol, and that in the feces mostly due to the unmetabolized drug. There was no indication of phenytoin N-glucoside being excreted in either the urine or feces of either subject, although one of the subjects was known to possess a particularly strong N-glucosidation capacity for barbiturates. The other subject was a poor metabolizer of debrisoquine and sparteine. Nevertheless, the DPH disappearance from serum and the DPH metabolite excretion in urine were virtually alike in these two subjects, indicating that the debrisoquine 4-hydroxylating and DPH hydroxylating capacities may be separable entities.

The fate of phenobarbitone in children in hypothermia and at normal body temperature.

Four critically injured children receiving large doses of phenobarbitone were studied during hypothermia (30 degrees - 31 degrees C) and at normal body temperature. The volume of distribution of phenobarbitone varied from 0.79 to 1.01 litres per kg and the serum t 1/2 ranged from 36.8 +/- 9.4 to 86.2 +/- 10.5 hrs. The percentage of dose recovered in urine in 16 days ranged from 40.5 to 65.5 per cent: 2.7 to 12.4 per cent as hydroxyphenobarbitone, 1.7 to 19.7 per cent as conjugated hydroxyphenobarbitone, 6.0 to 22.4 per cent as phenobarbitone-N-glucoside and 17.8 to 23.1 per cent as unchanged drug. After the body temperature was allowed to return to normal the rate of excretion of metabolites increased substantially and the rate of excretion of the unchanged drug decreased markedly. It is concluded that reduction in body temperature influences the volume of distribution, rate of metabolism and excretion of phenobarbitone.

Antipyrine metabolites in two populations.

After ingestion of 1 g antipyrine by healthy subjects of either Caucasian (n = 32) or Oriental (n = 18) background, urine was collected for 48 hr. and three major metabolites of antipyrine were determined. 21.8 +/- 1.1 (S.E.)% of the dose was excreted as 4-hydroxyantipyrine (4OHA), 12.7 +/- 0.6% as norantipyrine (NORA) and 5.9 +/- 0.3% as 3-hydroxy-methylantipyrine (3HMA). Oriental and Caucasian subjects showed similar mean recovery of each metabolite. The range of variation (F-test) of metabolite excretions between the 2 groups was again similar except for 3HMA which showed greater variation (p less than 0.01) in Orientals than in Caucasians. The fundamental similarity in antipyrine metabolism in the two populations is remarkable, because metabolite patterns of antipyrine differed significantly between subjects, and because the same two populations are known to differ much in their capacity for other drug oxidations.

Enzyme induction following a single dose of amobarbital in dogs.

The elimination of amobarbital in dogs was investigated by injecting various doses of amobarbital into a given animal. At low doses (3 mg/kg) serum levels declined in a first-order fashion. Superficially, at high doses (20 mg/kg) the relationship between serum concentration and time could be quantitatively characterized by simple one-compartment saturable kinetics. Indeed, qualitatively, saturation of the amobarbital-metabolizing enzymes was indicated by a shallower initial slope of the semilogarithmic concentration--time profile at the high than at the low dose. However, in addition, an acute enzyme induction phenomenon was observed which was indicated by a shorter terminal half-life of amobarbital at the high dose than after the low dose and also by a shortening in antipyrine half-life.

Seasonal variation of aryl hydrocarbon hydroxylase inducibility in human lymphocytes in culture.

The aryl hydrocarbon hydroxylase (AHH) inducibility in lymphocytes from peripheral blood of 92 healthy young subjects was tested during a period of 8 months (November - June). The average AHH inducibility was found to be higher from March to June than from November to February, indicating a seasonal variation. Various other features of lymphocytes in culture also appeared to change with the season.