Subzero preservation of mechanically prepared porcine islets of Langerhans: Response to a glucose challenge in vitro

Islets of Langerhans, prepared by a new mechanical process and avoiding enzymatic digestion were frozen to ?196 °C. Two rates of freezing were compared, instantaneous directly into liquid nitrogen and slow freezing at 1 °C min^?1. Post-thaw survival was greater after slow freezing.Three concentrations of dimethyl sulfoxide (DMSO) were compared. The 10% $\text{v}\text{v}$ concentration was found to give greater success than 20 or 30%. Contaminating exocrine tissue was found not to survive the freezing process.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Permeability of the human erythrocyte to glycerol in 1 and 2 m solutions at 0 or 20 °C

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation $\text{ds}\text{dt}\text{= P}{}_{\mathrm{<mtext>\gamma </mtext>}}\text{A}$[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of P_γ the permeability constant for the solute. Estimates of P_γ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of P_γ were about 2.5 × 10^?4 cm/min at 20 °C and about 0.9 × 10^?4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of P_γ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

Preliminary crystallographic data for beta-lactamase I from Bacillus cereus 569.

Crystals of β-lactamase I from Bacillus cereus 569 are monoclinic, space group C2 with unit cell dimensions $\text{a = 143·0 (± 0·5), b = 35·8 (± 0·1), c = 52·7 (± 0·2)}\text{A}\text{?}\text{, β = 97·0 (± 0·1) °}$, and one molecule of molecular weight about 28,000 per asymmetric unit.     

Preliminary crystallographic data for Azotobacter cytochrome c5.

Two closely related crystal forms of dimeric cytochrome c₅ from Azotobacter rinelandii have been grown. The crystals belong to space groups (C2 with $\text{a = 45·0, b = 38·4, c = 41·3}\text{A}\text{?}\text{and}\text{β = 101 ° 0′}$; and C1 (a centered triclinic cell) with $\text{a = 46·0, b = 37·6, c = 49·4}\text{A}\text{?}$, α = 87 ° 20′, β = 96 ° 40′ and γ = 90 ° 0′. In C2 the 24,000 molecular weight dimer lies on a Crystallographic 2-fold axis; in C1 the entire dimer occupies the asymmetric unit.     

Inhibition by dexamethasone of the in vitro transport of 3-O-methylglucose into rat thymocytes

Reduced glucose transport across the plasma membrane and reduced phosphorylation may both be responsible for the early inhibitory effect of physiological concentrations of glucocorticoids on glucose uptake by rat thymocytes.The early inhibitory effects of glucocorticoids (5 · 10^?7 M dexamethasone) on glucose consumption and ¹⁴CO₂ formation from d-[U-¹⁴C]glucose were reproduced.The total uptake curve of 4.8 μM $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ was biexponential with $\text{t}\text{1}\text{2}$ of 1.1 min and 36 min, respectively, the rapid part comprising about 50% of the equilibrated intracellular water space. The latency of the effect of 5 · 10^?7 M dexamethasone on $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ uptake ranged from 15 to 100 min and the inhibition varied from 15 to 55% independently of the lag period. The effect of 3-O-methylglucose concentration on the initial uptake by steroid-responsive cell preparations was tested after 45 min of preincubation with or without 5 · 10^?7 M dexamethasone. In 12 experiments dexamethasone reduced V from 1.36 ± 0.16 mmol · min^?1 · l^?1 cell water to 0.81 ± 0.10 mmol · min^?1 · l^?1 cell water with insignificant change of K_m (6.0 mM versus 5.9 mM). Dexamethasone had similar effect after 90 or 120 min.The variabilities of control cell transport capacity, the lag period and the magnitude of the dexamethasone effect could not be accounted for by changes in pH, effects of cell density, concentrations of albumin, ethanol, nucleosides, pyruvate or correlated to age and sex of the rats. In conclusion the inhibition of glucocorticoids on glucose consumption by thymocytes appears to be an inhibited plasma membrane transport capacity.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alcohol-induced potentiation of the neurally evoked contractions of the retractor unguis muscle of the locust, Schistocerca gregaria

Each of a series of seven monohydroxyl alcohols caused an increase in the force of the neurally evoked contractions of the innervated retractor unguis muscle isolated from the metathoracic femurs of the locust, Schistocerca gregaria. The negative logarithm of equipotent concentrations of the alcohols was proportional to the logarithm of the partition coefficient (1-octanol/water) of the alcohols: $\text{log}\text{1}\text{C}\text{= 1·282}\text{log}\text{P+1·684}$, where C is concentration, and P is the partition coefficient.Ethylene glycol and glycerol did not potentiate the contractions, possibly due to their very low lipophilic character. Acetone (10^?2 M) and benzene (10^?5 M) also potentiated the neurally evoked contractions of the insect muscle.     

On the possibility of the MN blood group determination in human bones

After a series of preliminary tests on inert substances and on saliva drawn from donors of know MN blood group, 31 right femura from Pisa cemetery buried for 25–30 years and 37 eneolithic femura from Gaudo necropolis near Paestum (2500 – 2000 B.C.) were submitted to 140 assays for the MN system using the standard technique already devised in our Laboratory for ABO blood group determination.Positive and reproducible results were obtained in 18:31 recent femura (58%) and in 24:37 eneolithic bones (65%). The following phenotype and gene frequencies were obtained:

$\text{recent femura:}\text{8.}\text{M}\text{+3.}\text{N}\text{+7.}\text{MN}\text{=18; 0·64m+0.·36n=1}$

$\text{eneolithic femura:}\text{9.}\text{M}\text{+6.}\text{N}\text{+9.}\text{MN}\text{=24; 0·56m+0·44n=1}$

When the ABO and the MN blood group determinations are performed in parallel, a significant positive connection between the two diagnosabilities is observable: P = 0·0387 in the case of the eneolithic bones, $\text{P ? 0·02}$ in the case of the total sample of 59 eneolithic and recent bones tested in parallel. This fact could be reasonably explained considering the similarity of the chemical structure of MN and ABO glycoproteins, which would account for a similar behaviour in preservation.The absence or the very low concentration of M and N substances in tissues and body fluids would on the other hand agree with the percentage of diagnosability: lower in the MN system (60–65%) than in the ABO system (75–80%). The more problematical character of MN blood group typing of bones could be better approached if further research were done.     

Correlation of the perceived texture of random coil polysaccharide solutions with objective parameters

A wide range of concentrated random coil polysaccharide solutions have been assessed for textural attributes by a trained sensory panel. The only textural terms invoked to describe these model systems were ‘thickness’ and ‘stickiness’, which were shown to be highly correlated, and essentially identical numerically, using a ratio scaling technique. Viscosity (η) measurements over a wide range of shear rates (γ) for all these samples gave flow curves (log η versus log γ) of the same form. Differences in flow behaviour between samples could then be characterised completely by two parameters, the maximum viscosity at low shear rates (η₀), and the shear rate ($\text{γ}\text{?}{}_{\mathrm{0·1}}$) at which $\text{η =}\text{solη}{}_0\text{10}$. A simple linear relationship was demonstrated between these two parameters and perceived thickness (T) or stickiness (S), irrespective of polysaccharide type. For Newtonian liquids, log T (or log S) varied linearly with log η. Hence the effective ‘in-mouth’ thickness of random coil polysaccharide solutions, in normal viscosity units, may be predicted directly from η₀ and $\text{γ}\text{?}{}_{\mathrm{0·1}}$ by the simple relationship: $\text{log}\text{η}{}_{\mathrm{N}}\text{= 1·13}\text{log}\text{η}{}_0\text{+ 0·45}\text{log}\text{γ}\text{?}{}_{\mathrm{0·1}}\text{? 1·72}$ where η_N is the viscosity of a Newtonian solution which would be perceived as identical in thickness (and stickiness) to the polysaccharide solution.     

Influence of various cooling and warming temperatures on survival after thawing of cryopreserved Plasmodium berghei

Heavy concentrations of viable P. berghei in the natural milieu [20% ($\text{v}\text{v}$) parasitized red blood cells, or 20% ($\text{w}\text{v}$) homogenate of splenic tissue in which malarial cells sequestered wer suspended in a serum-free, protective medium. Various rates of cooling are designated as low (1.3 °C/min) and intermediate (4 °C/ min) on exposure in cold gas evolving from liquid nitrogen refrigerant to ?70 °C, and this followed by direct immersion in the low temperature refrigerant (?196 °C). Cooling designated high was accomplished by abrupt immersion of the sealed vials with the live malaria-bearing tissue in the liquid nitrogen refrigerant. Rates of warming and thawing were designated low (after slow rewarming of frozen tissue in air at 25.5 °C) and high (after rapid rewarming and thawing in a water bath at 40 °C). Strip chart recordings were made of the complete cooling and freezing wave patterns of the suspending medium to ?70 ° C. The functional survivals of the freeze-thaw P. berghei malaria were measured by a special infectivity titration method.None of the cooling and freezing treatments adversely influenced the parasite survivals. Our data showed the optimum cooling velocity that maximally protected this highly lethal P. berghei strain within the host erythrocyte matrix was 1.3 ° C/min to ?70 to ?196 ° C. The functional survivals of two RBC stabilates with P. berghei, after retrieval from 25 days storage in the liquid nitrogen refrigerant, excelled by more than 100-fold the infectivity titer found by viability assay in the pool of the 0-days nonfrozen infected RBC.The precise factors favoring the maximal survivals of the freeze-thaw P. berghei are unclear. Several factors, singly or in combination, may have played key roles in protecting the living P. berghei from the freeze-thaw damage. These factors are: The composition of the suspending medium fortified by additions of bicarbonate, glucose, lactalbumin hydrolysate and yeastolate; the presence of naturally occurring peptide-containing materials surrounding the parasites in the host red cell milieu; and the protective glycerol agent. Any of these constituents singly or combined possess potential for reducing freeze-thaw injury to the parasites to produce maximal survivals.     

Cryoprotection in hearts as measured by CPK

Rat hearts were treated with a cryoprotectant solution composed of glycerol and DMSO in concentrations ranging from 10–25% $\text{v}\text{v}$, and then frozen in liquid nitrogen. Creatine phosphokinase activity was then measured spectrophotometrically and activities were compared with activities of frozen-untreated hearts, unfrozen-treated hearts, and unfrozen-untreated hearts. Independently freezing or treating increased enzyme activity, but when hearts were treated and then frozen, activity diminished below that of the unfrozen-untreated group. Evidence suggests (1) DMSO-glycerol solution either has a toxic effect that increases with concentration and freezing additionally aggravates this effect, or the concentrations of cryoprotectants used do not adequately protect the tissue during freezing which causes damage to the contractile mechanism; and (2) the anoxic state of the tissue causes depletion of ATP and creatine phosphate such that creatine phosphokinase is inactive or not able to function.     

Thermoelectric (seebeck) effect of the cuticle of social wasps

The thermoelectric (Seebeck) coefficient ($\text{S =}\text{ΔV}\text{ΔT}$) in various cuticular areas of the Oriental hornet (Vespa orientalis) fluctuates from 0·3 to 2·4 mV deg^?1 within a temperature range of 27–36°C and when the temperature difference between the two measuring electrodes (ΔT) is 0·6–8·0°C. The values measured on the brown-colored cuticle suggest an n-type conduction, while those measured on the yellow-colored cuticle point to a p-type conduction. It is suggested hornets use this phenomenon for temperature detection.     

An attempt to isolate Brucellaabortus from uterine flushings of brucellosis-reactor donor cattle

Two experiments were conducted to test for the recovery of brucella organisms from uterine flushings and harvested embryos of sero-positive embryo donor females. In Experiment I, 16 sero-positive cows were superovulated with FSH treatments and artificially inseminated at 12, 24 and 36 hours following the onset of estrus with brucella-free semen. At 48 hours after the onset of estrus, one half the potential donor females were administered an intrauterine inoculation of 3.3 to 4.6 × 10⁴$\text{Brucella}$$\text{abortus}$ (strain 2308) organisms while the remainder received a control inoculation. In Experiment II, the same 16 cows were similarly administered superovulatory treatments and inseminated following estrus. The uterine inoculation was increased to 1.5 to 2.5 × 10⁸ organisms administered 48 hours following estrus. Samples of recovered flushing medium and homogenized embryo residues were placed into a validated $\text{in}$$\text{vitro}$ culture system to detect the presence of brucella bacteria. Uterine flushings and embryos recovered from 31 females exhibiting estrus following FSH treatments were free from either field strain or the inoculated $\text{B.}$$\text{abortus}$ (strain 2308) contamination. The flushings obtained from a single female, which did not respond with estrus following FSH treatment but was inoculated at appointment, did contain $\text{B.}$$\text{abortus}$ which was identified as the inoculated strain 2308 and not field strain organisms. These results indicate that brucella contamination of flushing media and harvested embryos will not likely be incurred when collecting embryos from sero-positive donor females. These findings offer further encouragement for the use of embryo transplantation as a method to produce brucella-free offspring from infected cows.     

Stimulation of caffeine metabolism in the rat by 3-methylcholanthrene

Groups of adult male rats treated with 3-methylcholanthrene, phenobarbital or vehicles alone, were administered caffeine either orally or intravenously. Serum caffeine concentrations were measured by radioimmunoassay. In vehicle and phenobarbital pretreated animals, caffeine elimination kinetics were non-linear. In control animals, the $\text{in}$$\text{vivo}$ apparent Km was 8 μg·ml^?1 (40 μM) and the apparent Vmax was 0.1 μg·ml^?1·min^?1 (0.5 μM·min^?1). Phenobarbital pretreatment did not change the apparent Km but slightly increased the apparent Vmax. 3-Methylcholanthrene pretreatment dramatically altered the elimination kinetics of caffeine, whether caffeine was given orally or intravenously. The elimination of caffeine from serum of 3-methylcholanthrene pretreated rats was first order with a $\text{t}\text{1}\text{2}$ of approximately 14 minutes.Our results are consistent with the proposed involvement of the cytochromes P-450 monooxygenase system in the elimination of caffeine. In addition, our results suggest that caffeine is a moderately poor substrate for the cytochromes P-450 present in control and phenobarbital-pretreated rats, but a particularly good substrate for the form(s) induced by 3-methylcholanthrene.