D. Kadar

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.

A Sudden death associated with the use of sodium nitroprusside for induction of hypotension during anaesthesia

SummaryA death from cyanide poisoning during operation was attributable to the use of approximately 400 mg (10 mg per Kg) sodium nitroprusside as an hypotensive agent. Autopsy cyanide levels of 0.5 mg per cent in blood and 0.3 mg per cent in urine and thiocyanate levels too low to measure in either, suggest an abnormality of cyanide metabolism. The lethal dose of sodium nitroprusside appears to be 7 mg per Kg. A safe regimen would be 3.5 mg per Kg as the anticipated total dose for the period of hypotension. Patients resistant to the full hypotensive effects of sodium nitroprusside, or unable to maintain the level of hypotension, or requiring more than 3.5 mg per Kg to achieve the required effect for the anticipated time, may accumulate toxic levels of cyanide in the blood.Patients with Leber’s optic atrophy, tobacco amblyopia and possibly those with other nerve atrophies should not receive sodium nitroprusside or any other cyanide precursor.RésuméLes auteurs rapportent un décès per-opératoire par empoisonnement au cyanure chez un malade ayant reçu une dose totale de 400 mg de Nitroprussiate de sodium (10 mg/kg). La détermination du taux sanguin et du taux urinaire de cyanure à l’autopsie a montré des taux respectifs de 0.5 et 0.3 mg%. Des taux de thiocyanates trop bas pour être déterminés suggèrent un métabolisme anormal du thiocyanate.La dose léthale de Nitroprussiate semble se situer autour de 7 mg/kg. L’établissement anticipé d’une dose totale plus petite que 3.5 mg/kg pour toute la durée de l’intervention nous semble recommandable.Chez des patients qui présentent une résistance anormale aux doses habituelles de Nitroprussiate ou incapables de maintenir leur niveau d’hypotension avec une dose constante de Nitroprussiate ou qui requièrent une dose totale anticipée supérieure à 3.5 mg/kg, le taux de cyanure sanguin peut atteindre des seuils toxiques.Les malades présentant le syndrome d’atrophie optique de Leber, une amblyopie tabagique et possiblement ceux souffrant d’autres atrophies nerveuses ne devraient pas recevoir de Nitroprussiate.

Comparative drug elimination capacity in man-glutethimide, amobarbital, antipyrine, and sulfinpyrazone.

The apparent elimination half‐lifes were determined for 4 drugs that undergo hydroxylation in men. Glutethimide (500 mg), amobarbital sodium (130 mg), antipyrine (1.0 gm), and sulfinpyrazone (400 mg) were each taken orally by 10 healthy men at weekly intervals. When the half‐lifes in these 10 individuals were compared, positive correlations were found among 3 out of 4 drugs: glutethimide‐amobarbital (r 0.69, p < 0.05), glutethimide‐sulfinpyrazone (r 0.64, p < 0.05), and amobarbital‐sulfinpyrazone (r 0.87, p < 0.01). If these data are generally valid, it would mean, for example, that one could, from the sulfinpyrazone half‐life in a given subject, predict the amobarbital half‐life of the same individual within about ± 7 hours (30% of average half‐life, while the interindividual differences could be 300%). Antipyrine half‐life was not correlated with the half‐life of any of the other 3 drugs. The urinary levels of 6β‐hydroxycortisol were also determined in the same sub;ects and compared with the half‐lifes of the 4 drugs. The correlation coefficients taken singly were not statistically significant, but they were all negative and collectively seem to indicate an expected trend.

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 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‐(β‐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 2 metabolite concentrations were the same in both populations. 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 (t½) were established by calculation. A Caucasian female with no capacity for N‐glucosidation was found during the first part of this population survey.16 An Oriental male with only trace capacity for amobarbital hydroxylation was found in the second part.

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°–31°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 té 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 pecrent: 2.7 to 12.4 percent as hydroxyphenobarbitone, 1.7 to 19.7 percent as conjugated hydroxyphenobarbitone, 6.0 to 22.4 percent as phenobarbitone-N-glucoside and 17.8 to 23.1 percent 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.RéSUMéOn a étudié quatre jeunes blessés graves recevant du phenobarbitone à hautes doses en hypothermie (30°–31°C) et à température normale. Le volume de distribution du phénobarbitone a varié de 0.79 à 1.01 litre par kg et la t1/2 sérique s’est située entre 36.8 ± 9.4 et 86.2 ± 10.5 heures. Le pourcentage de la dose recouvrée dans l’urine en 16 jours a été de 40.5 à 65.5 pour cent: 2.7 à 12.4 pour cent sous forme d’hydroxyphenobarbitone, de 1.7 à 19.7 pour cent sous forme d’hydroxyphenobarbitone conjugué, de 6.0 à 22.4 pour cent sous forme de phenobarbitone-N-glucoside et de 17.8 à 23.1 pour cent sous forme inchangée. Lorsqu’on a laissé la température revenir à la normale, la vitesse d’excrétion des métabolites a augmenté substantiellement alors que l’excrétion sous forme inchangée a subit une baisse importante. On en conclut que la baisse de température de l’organisme a influencé le volume de distribution, la vitesse du métabolisme et de l’excrétion du phenobarbitone.

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 individuals 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 persons 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‐β‐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 < 0.02).

Isoproterenol metabolism in children after intravenous administration

The metabolism of intravenously administered isoproterenol was investigated in children with severe brain damage between the ages of 6 and 16, using isoproterenol‐7‐3H. The serum concentration of .9H declined in a biphasic manner with the half‐lives of 2.5 to 5 minutes estimated from the rapid first phase and 3 to 7 hours estimated from the second slower phase. The drug is rapidly metabolized and after 15 to 30 minutes less than 10% of the .1H in the serum represents unchanged drug and after 1 hour less than 3%. The metabolites found in the serum and urine are free and con;ugated 3‐0‐methylisoproterenol, con;ugated isoproterenol, and an unknown substance that is excreted mainly in the bile. About 4% of the dose was eliminated in the urine in 5 minutes, 60% in 6 hours, and over 90% in 24 hours. Less than 15% of the urinary radioactivity represented unchanged drug, and most of that was eliminated during the first 2 hours.

A method for measuring glutethimide (doriden®) in human serum after intake of therapeutic doses

Abstract A method is described that permits the determination of 0.25 μm/ml of glutethimide in 0.5 ml of serum. Because of this high sensitivity, the method is suitable for pharmacogenetic studies and for tests of bioavailability. A crucial part of the method is the extraction, purification and concentration procedure, which requires multiple steps in order to eliminate endogenous substances and possible metabolites of the drug that tend to interfere with measurements performed by gas—liquid chromatography.