Changes in theophylline metabolism during postnatal development in rat liver slices.

The metabolism of theophylline was studied in liver slices of young and adult rats. Theophylline and six metabolite fractions were recognized in adult liver by thin-layer radiochromatography and high performance liquid chromatography: 1-methyluric acid; 1-methylxanthine; 1,3-dimethyluric acid and/or 3-methylxanthine; caffeine; a uracil derivative and two unknown polar compounds. Preincubation with caffeine or theobromine inhibited theophylline metabolism. Allopurinol decreased the formation of three metabolite fractions but markedly increased the production of 1-methylxanthine. SKF 525-A inhibited the overall metabolism of theophylline. The specific activity of the enzyme system was 3.2 +/- 0.4 nmoles X (g liver)-1 X hr-1 in the 4- to 5-day-old rat and increased to a peak of 25.7 +/- 1.7 in the 28-day-old; values for Km and Vmax in the 7- and 28-day-olds were 132.1 and 67.5 microM, and 23.9 and 52.1 nmoles X (g liver)-1 X hr-1 respectively. Theophylline and the same six metabolites were identified in young and adult rats, but the development pattern was not uniform. Peak age-related activity and involvement of mixed-function oxidase system are features which are common to theophylline and caffeine metabolism. Xanthine oxidase played a role in theophylline metabolism. Formation of caffeine from theophylline was not dependent on a lack of activity of other pathways.

Tissue distribution of caffeine in premature infants and in newborn and adult dogs.

Caffeine concentrations were measured in postmortem tissues of two premature infants who had received caffeine for the prevention of apnea. The distribution of caffeine was not uniform: Up to a 2.1-fold difference in caffeine concentrations existed among the various tissues in one infant and up tu a 4.3-fold difference in the other. This variability was only slightly reduced when caffeine concentrations were corrected for the water content of the respective tissues. Caffeine was also analyzed in tissues of newborn and adult dogs 3, 10, and 36 hr after single doses of caffeine (50 mg/kg IV). The decrease in caffeine concentrations was slower in the puppy than in the adult dog. The ratio tissue water caffeine/plasma water caffeine decreased with time after administration of this drug: It ranged between 0.9 and 1.1 at 3 hr and between 0.5 and 1.0 at 10 hr in adult dogs, and it varied from 0.8 to 1.3 at 3 hr and from 0.5 to 0.8 at 36 hr in puppies. Greater caffeine concentrations in newborn than in adult tissues following a given dose are consistent with the known slow elimination rate of caffeine in the young organism.

Comparative toxicity of caffeine and aminophylline (theophylline ethylenediamine) in young and adult rats.

The toxicity of aminophylline and caffeine was studied in adult and 2-day-old rats following a single subcutaneous injection of the respective drug. Following the injection of high doses of either methylxanthine, adult rats developed convulsions, tremors, lethargy and licking of lips. In adult rats, the LD50 of caffeine and aminophylline was the same after 24 h and after 1 week of observation: caffeine 265 mg/kg, and aminophylline 202 mg/kg (theophylline base 172 mg/kg). In young rats, the LD50 was greater when the observation was carried out for 1 week than at 24 h after the injection; at 24 h: caffeine 220 mg/kg, and aminophylline 169 mg/kg (theophylline base 144 mg/kg); at 1 week: caffeine 155 mg/kg, and aminophylline 140 mg/kg (theophylline base 119 mg/kg). Young rats failed to gain weight at a normal rate after administration of either methylxanthine. The greater toxicity of both methylxanthines in newborn animals may be at least partly due to the extremely slow elimination of theophylline and caffeine in the neonate.

Caffeine pharmacokinetics in young and adult dogs.

The pharmacokinetics of caffeine were studied in young (age: 1 day, 7 days, 14 days, 30-45 days) and adult dogs following a single intravenous dose of 50 mg/kg. Mean (+/-SE) plasma elimination half life (T1/2) was 47.5 (+/-5.35) h in 1-day-old puppies, as opposed to 6.66(+/-0.85) h in adult dogs. A rapid decrease in plasma T1/2 values occurred during the first 2 weeks of life. At about 14 days of age caffeine plasma T1/2 was similar to that of adults. The volume of distribution was greatest (0.94+/- 0.03 liters/kg) and the total body clearance was smaller (2.81 +/- 0.73 liters/kg/min X 10(-4)) in the 1-day-old animals. The much smaller body clearance of caffeine in the newborn as compared to the adult dog could be due to a lower rate of metabolism and/or renal excretion of this drug in the young.

Urinary metabolites of caffeine in young dogs.

Urinary elimination of [1-methyl-14C]caffeine was investigated in young dogs. Mongrel dogs aged 2 days, 1 week, and 5 weeks received single doses of caffeine (50 mg/kg) through an orogastric tube. Eight dogs, each studied once, were involved. At identical times after the dose the ratio of urinary caffeine metabolites to unchanged caffeine was greatest in the 5-week-old and smallest in the 2-day-old puppy; cumulative ratios at (or very close to) the plateau of urinary excretion varied between 8.7 and 17.8 in the 5-week-old, 3.6 and 3.8 in the 1-week-old, and 2.2 and 2.4 in the 2-day-old dogs. The time needed to reach the plateau of the cumulative excretion of radioactivity in the urine decreased with age. Initially only caffeine was detected in the urine of the 2-day-old and 1-week-old dogs; the first quantitatively important metabolites were trimethyluric acid and 6-amino-5-(N-methylformylamino)-1,3 dimethyluracil (also known as 1,3,7-trimethyldihydrouric acid) in the 2-day-old, and the uracil derivative and theophylline in the 1-week-old and the 5-week-old puppies. The percentage of demethylated uric acid metabolites and uracil derivatives increased progressively with increasing age; this indicates increased demethylation, oxidation to uric acids and hydrolysis of the imidazole ring of caffeine with increasing age. These results are consistent with the previously reported slow plasma elimination of caffeine in the newborn and confirm the limited capacity of the young (as compared to the adult) mammal to metabolize caffeine.

Cardiac toxicity of digoxin in newborn and adult rats.

We investigated the age-specific arrhythmogenic effects of digoxin in the rat. Adult female rats (n = 26) and one-day-old newborns (n = 20) were anesthetized with pentobarbital and injected subcutaneously with varying doses of digoxin. Electrocardiograms (ECG) were monitored continuously for four and one-half hours following digoxin administration. The arrhythmogenic dose 50 (AD50) and lethal dose 50 under anesthesia (LD50) were determined using the method of Litchfield and Wilcoxon. AD50 in adults was 13.0 +/- 1.0 mg/kg (X +/- SD) compared with 2.9 +/- 0.3 mg/kg in the newborns (P less than 0.01), and LD50 in adults was 30.0 +/- 1.9 mg/kg compared with 5.0 +/- 0.2 mg/kg in the newborns (P less than 0.01). Arrhythmias appeared earlier in adults (54 +/- 11.5 minutes after digoxin, X +/- SEM) than in newborns (132.2 +/- 11.0 minutes, P less than 0.01). Paroxysmal atrial tachycardia was the predominant arrhythmia in adults (73%), while transient sinus bradycardia appeared in only 9%. In contrast, all newborns with arrhythmias had severe sinus bradycardia and 69% had profound first degree heart block as well. In conclusion, the newborn rat is more sensitive to digoxin toxicity and develops lethal arrhythmias much more readily than the adult.

Recent duplication and germ-line diversification of rat immunoglobulin kappa chain gene joining segments.

Sequence determination of the joining segment gene (J) cluster in the kappa chain (J kappa) in the embryonic context demonstrates that rat genome contains seven J kappa gene segments that expanded from an ancestral cluster of five J kappa genes. The rat J segments are separated by about 300 base pairs (bp) and are flanked 5' by the presumed variable region (V)/J recombination signal sequence and 3' by the RNA splicing signal. Two of the J gene segments designated J2A and J2B and their 5'-flanking spacer DNA bear striking homology to J2 and its 5'-flanking spacer. Thus, the unit of duplication was the entire J kappa coding region and 5' noncoding spacer (345 bp). The duplication probably occurred as two separate unequal crossing-over (UXO) events. The first UXO event can be confined to recombination within an identical stretch (14 bp long) located at the 3' ends of the coding regions of J1 and J2. The second event could involve a longer segment (372 bp) of tight homology generated by the first UXO event, thus increasing the probability of repeated expansion of the same DNA segment. The sequence homology among the rat duplicated segments (98-99%) is larger than the homology between the corresponding rat and mouse segments (89%), showing that the rat J kappa gene expansion must have occurred after rat and mouse divergence 10 X 10(6) yr ago. We estimate that the first and second UXO events occurred 2 X 10(6) and 1 X 10(6) yr ago, respectively. J3 of rat and mouse share the same mutation (G leads to C) in the RNA splicing signal that presumably inactivates J3. This mutation preceded divergence of the two species. A mutation in the first nucleotide of codon 96 has occurred in both duplicated segments, the only position along 345 bp where J2, J2A, and J2B differ from each other. This results in three different amino acids at position 96 not present in any other J kappa. These mutations are physiologically significant because they diversify the third complementarity-determining region (CDR3) and, thus, may reflect selective pressure to increase antibody diversity. The germ-line diversification of CDR3 was exercised within the last 1-2 X 10(6) yr.