of chocolate consumption to assess general health of the subjects and any effects from subacute chocolate consumption. Background methylxanthine ...
HIGH LEVELS OF METHYLXANTHINES IN CHOCOLATE DO NOT ALTER THEOBROMINE DISPOSITION CAROL A. SHIVELY, MS., STANLEY M. TARKA, JR., Ph.D., MAURICE J. ARNAUD. Ph.D.. BARRY H. DVORCHIK; ph.D.; G. THOMAS PASSANANTI, Ph.D., and ELLIOT S. VESELL, M.D. Hershey and Spring House, Pa., and LaTour-dePeilz, Switzerland From the Life Sciences Division. Hershev Fwds Corporation Technical Center and the DepaAment of Pharmacolo The Pennsylvania State Universit College of h&icine, Hershev: McNeil Pharmaceuticar Drug-Metabolism Department, Spring House and the Research Department, Nestle Products ‘fechnical Assistance Co., Ltd., LaTour-depeilz.
Reprinted from CLINICAL PHARMACOLOGY AND THERAPEUTICS, St. Louis
Vol. 37, No. 4, pp. 415424, April, 1985, (Copyright @ 1985, by The C.V. Mosby Company) (Pnnted in the U.S.A.)
High levels of methvixanthines in chgcolate . . do not al& theobromine disposition
.'I> __
Theobromine disposition was measured twice in 12 n o d men, once after 14 days of abstention from ail methyixanthines and once after 1week of theobromine (6 mg/kg/day) in the form of dark chocolate. Mean theobromine ty2, apparent volume of distribution, and clearance after abstinence from ail methylxanthines were 10.0 hours, 0.76 Líkg, and 0.88 ml/min/kg. High daily doses of chocolate for 1 week did not change these values. After subjects abstained from methyixanthines, urinary radioactivis. over 72 hours after a single, oral dose of [8-"C]theobromine consisted of 42% 7-methykanthine, 20% 3-methylxanthine, 18% theobromine, 10Yo 7-methyluric acid, and 10% 6-amino-5[N-methylformylamino]-1-methyluracil.A week of daily theobromine consumption in the form of dark chocolate also did not alter this urinary profle of theobromine and its metabolites. Although these results might appear to M e r 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 bioavailabitity 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 M e r when that chemical is given in the pure compared with the dietary form. (CLINPHARMACOL"ER 37415-424,1985.)
Carol A. Shively, M.S., Stanley M. Tarka, Jr., Ph.D., Maurice J. Arnaud, Ph.D., Barry H. Dvorchik, Ph.D., G . Thomas Passananti, Ph.D., and Elliot S. Vesell, M.D. Hersbey and Spring House, Pa., and LaTour-de-Pe&, Switzerland
.*
Theobromine (3,7-dimethylxanthine) is related to two other common methylxanthines, caffeine (1,3,7trimethylxanthine) and theophylline (1,3-dimethylxanthine). Many people ingest methylxanthines in their diet well as in medications. The disposition of these compounds has clinical relevance not only because methylxanthines in the diet can stimulate the central nervous and cardiovascular systems, but also because dietary methylxanthines have been reported to inhibit theophylline metabolism, thereby diminishing theophylline's pharmacologic effects.I5The potential of all methylxanFrom the Life Sciences Division, Hershey Foods Corporation Technical Center and the Department of Pharmacology, The Pennsylvania State University College of Medicine, Hershey; McNeil Pharmaceutical, Drug Metabolism Department, Spring House; and the Research Department, Nestle Products Technical Assistance Co., Ltd., LaTour-de-Peilz. Supported in part by The Chocolate Manufacturers Association of the United States. Received for publication Sept. 21, 1984; accepted Dec. 7, 1984. Reprint requests to: Dr. Stanley M. Tarka, Jr., Life Sciences Division, Hershey Foods Corporation Technical Center, 1025 Reese Ave., Hershey, PA 17033-0805.
thines to exert pharmacologic effects, the potency of which differs among the methylxanthines, is indicated by their prominent treatment in almost every pharmacology text. Nevertheless, the precise mechanisms of their effects remain unclear, but may involve competitive inhibition of phosphodiesterase at high doses and antagonism of endogenous adenosine at lower doses. Since these effects and interactions largely depend on the concentrations of methylxanthines at their sites of action and metabolism, respectively, factors that influence systemic methylxanthine concentrations, such as bioavailability from dietary sources or inhibition of their biotransformation to inactive metabolites, are of potential clinical significance. observed that theobromine clearDrÖuillard et ance was reduced after five daily doses of an oral aqueous suspension of theobromine, 6 mgikg. The effect was reversed after 4 days of abstention from all methylxanthines. Another study reported that theobromine (200 mg theobromine in hard gelatin capsules every 8 hours for 4 days) did not change theobromine ~1earance.I~ Monks et al.I5 reported inhibition of [8-
415
416 Shively et al. ''C]theophylline elimination after caffeine (300 to 700 mg/day) with theophylline (125 mg/day) in tablet form in amounts equivalent to the usual dietary intakes of their subjects. Theophylline elimination was more rapid when subjects followed a methylxanthine-free diet.I5 While not prescribed for therapeutic purposes, theobromine is commonly ingested in various foods. We therefore examined whether theobromine in the form of chocolate can also inhibit its own elimination. To model high dietary theobromine intake (6 mglkglday), we chose a 7-day period of daily consumption of dark chocolate. Furthermore, because other dietary constituents such as protein and carbohydrate can alter methylxanthine elimination rates in man,9~'2~18 the diets of our subjects were standardized to avoid effects from such interference.
METHODS
I
[8-'4C]Theobromine (specific activity 7.8 mCi/ m o l ; radio chemical purity >99%) was synthesized by the Radiochemical Center (Amersham Corp.). Theobromine sodium acetate was obtained from Mallinckrodt Inc. The reference standards used in the HPLC analyses were theobromine (Sigma Chemical Co.), 3,7dimethyluric acid (Adams Chemical Co.), 3-methylxanthine, 3-methyluric acid, 7-methylxanthine and 7-methyluric acid (John Fluka Manufacturing Co.), and 6-amino-5 [N-methylformylamino] - 1- m e t h y h a cil. * The internal standard ß-hydroxyethyltheophylline came from Sigma Chemical Co. Samples of a European dark chocolate were provided by Hershey Foods Corporation and were analyzed for theobromine and caffeine content before ingestion. Our subjects were 12 healthy, nonsmoking, nonmedicated men 22 to 29 years old. As determined by previous dietary history, they were not heavy coffee, tea, or chocolate consumers. They refrained from all methylxanthine-containing foods and ate only standardized meals (2500 calories; 45% carbohydrate, 15% protein, and 40% fat) provided by the dietary department of the Milton S . Hershey Medical Center during the study. As a screen for dietary compliance, two morning urine samples collected during the 14 days of methylxanthine abstinence were analyzed for background contamination from theobromine and caffeine. Neither over-the-counter nor prescription drugs were taken for 2 weeks before or during the study. At 9 AM after an overnight fast, each subject received on two separate occasions an oral dose of 10 *Synthesized by Dr. Maurice Shamma of The Pennsylvania State University.
CLIN PHARMACOL THER APRIL 1985
mg/kg theobromine sodium acetate (equivalent to 6 mg/kg theobromine free base) with 60 yCi [8''C]theobromine. The first dose followed 14 days of abstention from all methylxanthines, and the second dose followed 7 consecutive daily doses of 6 mg/kg theobromine (as dark chocolate) at 9 AM each day. Solutions of theobromine sodium acetate were prepared in distilled water. Previous reports demonstrated virtually complete bioavailability of theobromine taken as an oral solution.' Blood specimens (5 mi) were drawn O, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after theobromine ingestion. Serum was obtained by centrifugation and was frozen at - 20" C until assayed for theobromine. Urine was collected into glass bottles containing 2 mi toluene as a perservative at O, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hours after theobromine ingestion. Urine volumes were recorded for each time point; i \ portion of the urine was frozen at - 20" C until assayed for theobromine and its metabolites. To estimate fecal excretion of theobromine, complete fecal samples were collected from two subjects (M. D. and D. S . ) over the periods O to 24 and 24 to 48 hours after theobromine. Clinical chemistry profiles (12 parameters) were determined in all subjects before and after the 7-day period of chocolate consumption to assess general health of the subjects and any effects from subacute chocolate consumption. Background methylxanthine (theobromine and caffeine) concentrations were determined in the morning urine samples from each subject by a method developed in our laboratory. Urine samples (100 pl) were aspirated through C-18 minicolumns with the Baker Solid Phase Extraction System (J. T. Baker Chemical Co.). Columns were then washed with distilled water before elution with HPLC grade methanol. After the eluent wai: dried under nitrogen, the residue was reconstituted witl. - 1I mobile phase (25% methanol in 0.5% acetic acid [v/v]) and injected onto the HPLC system. Theobromine and caffeine were quantitated by the peak area ratio by use of the intemal standard method of calibration. Theobromine concentrations in serum samples were measured by the method of Aldridge et al.' A serum aliquot (100 pl) was vortexed with 50 pi saturated ammonium sulfate. After addition of the internal standard solution (3 ml ß-hydroxyethyltheophylline, 1 pg/ ml, in chloroform/isopropanol [85 : 15; v/v]), the mixture was extracted for 20 minutes on a horizontal shaker and centrifuged for 5 minutes at 1500 X g. The aqueous layer was aspirated and the organic layer was transferred to another test tube and dried under nitrogen in a 45" C water bath. Immediately before injection, the residue was reconstituted in 400 p1 mobile phase
VOLUME 37 NUMBER 4
Methylxanthine efects on theobromine metabolism 417
Table I. Theobromine kinetics after a 6 mg/kg oral theobromine dose during methylxanthine abstinence or consumption Body weight (kg)
Dose* (mg)
79.0 88.5 59.0 77.0 80.5 78.5 70.5 69.0 69.0 74.5 84.5 85.0
474 53 1 354 462 483 47 1 423 414 414 447 507 510
9.0 8.2 10.0 11.8 9.8 8.6 9.2 9.8 9.7 10.3 10.8 13.3
O. 077 0.085 0.069 0.059 0.070 0.080 0.075 0.071 0.072 0.067 0.064 0.052
118.7 108.6 117.9 177.9 120.3 93.3 93.9 102.7 113.3 106.1 108.3 135.6
0.66 0.65 0.74 0.57 0.71 0.80 0.85 0.82 0.74 0.84 0.87 0.85
O. 84 0.92 0.85 0.56 0.83 1.O7 1.O6 0.97 0.88 0.94 0.92 0.74
76.2 k8.3
45 8 f50
10.0 21.4
0.070 k0.009
116.4 k22.6
0.76 I 10
0.88 *O. 14
M. M. H.M. R. M. T. S. R. S. I. s. D. S .
79.0 88.5 59.0 77.0 80.5 78.5 70.5 69.0 69.0 74.5 84.5 85.0
474 53 1 354 462 483 47 1 423 414 414 447 507 510
8.1 7.6 10.5 9.9 10.3 7.5 8.2 9.9 11.8 10.3 10.1 12.7
0.086 0.091 0.066 0.070 0.068 0.092 0.085 0.070 0.059 0.067 0.068 0.055
107.3 102.0 142.3 162.1 127.7 78.4 84.0 101.7 110.1 120.8 110.9 147.7
0.65 0.65 0.64 0.53 0.69 0.83 0.84 0.84 0.92 0.74 0.80 0.74
0.93 0.98 0.70 0.62 0.78 1.28 1.19 0.98 0.91 0.83 0.90 0.68
76.2 k8.3
45 8
9.7
I SD
k 50
f 1.6
0.073 20.012
116.2 +-25.2
0.74 20.11
0.90 10.20
Subject
MX Abstinence M. D. S. H. D. I. K. K. T. K. M. M.
H.M. R. M.
T.S. R. S. b.SS. Xt fSD MX Consumption M. D. S . H. D. I. K. K.
T.K.
xt
t%
ß
(hr)
(hr-')
AUCO.,
(mgJL . hrj
aVd (LJkg)
o.
CI (mllminlkg)
MX = Methylxanthine. *The amount of theobromine (6 mgikg free base) present in the administered dose of 10 mgikg theobromine sodium acetate. tThere were no statistically significant differences (P S 0.05; paired t test).
(10% acetonitrile in 0.5% acetic acid [v/v]). Theobromine and caffeine were quantitated by the peak area ratio with use of the internal standard method of calibration. An additional aliquot (250 pl) of each serum sample was analyzed by liquid scintillation counting to determine total radioactivity at each time point. Previous work in our l a b ~ r a t o r ydemonstrated '~ that total serum radioactivity can be entirely accounted for by theobromine. Urine samples were prepared for HPLC analysis by filtration through 0.45 p syringe filters. Samples were then injected onto an HPLC gradient system for quantitation of [8-'4C]theobromine and its metabolites by means of an HPLC radioactivity monitor with a solid scintillator flow-cell detector. A chromatogram of a
standard solution of theobromine and its principal metabolites was used to identify peaks on the radioactivity monitor. Separation was achieved on an Ultrasphere (5 p) ODS column (4.6 mm id x 30 cm; Altex Scientific Inc.) at a flow rate of 1 ml/min, with an initial mobile phase composition of 3% acetonitrile in 0.5% aqueous acetic acid and a final composition of 9% acetonitrile in 0.5% aqueous acetic acid (v/v). The concave elution program No. 9 on the Model 680 Automatic Gradient Controller (Waters Associates Inc .) generated the gradient. Final solvent composition was reached in 26 minutes. Conversion of counts per minute from the radioactivity monitor to disintegrations per minute was by the formula: Z = E . (v'ív) (Q - B), where Z is the counts per minute, E is the efficiency determined by
CLIN PHARMACOL THER APRIL 1985
418 Shiveíy et al.
r
r
r
MD
10
5
o Aner 7 days of chocolate consumption
0.5
20
r
r
c
10.0 hr
r
MM
c
Aner 14 days DIMX abstinence
TK
c
9.8 hr 7.5 hr
0.5
I
I
l
I
I
1
I
r
2o
t , , , , , ,t r r 9.8 hr
0.5
I\ tt
1
9.7 hr
r
RS
10
\ ; 5
o
10.3 hr
!\
10.3 hr
-
-
10.1 hr
+ !
12.7 hr
13.3 hr 1 -
0.5 -
10.8 hr
I
l
I
I
I
I
-
1
I
l
I
I
J
I
I
i
l
Hours after Theobromine Administration Fig. 1. Theobromine serum concentration decay curves after 14 days of abstinence from methylxanthines or 7 days of chocolate consumption. Theobromine sodium acetate was taken as a single, oral dose equivalent to 6 mg/kg theobromine as free base.
I
I
VOLUME 37 NUMBER 4
Methylxanthine effects on theobromine metabolism 419
Table II. Theobromine t % and AUC derived from serum [8-'4C]theombromine concentration MX Abstinence
M. D. S . H. D. I.
K.K. T. K. M.M. H. M. R. M. T. S. R. S . I. s. D. S.
.-->
AUC (mgIL . hr)
t% fhr)
Subject
z*
+I SD
MX Consumption
AUC (mgIL . hr)
t% fhr)
9.2 7.4 10.4 12.0 10.1 8.1 7.5 9.6 10.9 11.2 9.2 12.5
124.5 120.3 132.6 192.2 155.5 127.2 123.0 133.4 137.1 151.8 146.0 173.4
8.2 7.1 10.3 10.9 11.0 7.5 8.8 10.2 11.6 11.2 10.3 13.4
9.8
143.1 k22.0
10.1
138.7
k 1.8
2 24.0
k 1.6
112.0 108.1 137.6 163.7 163.9 105.4 117.0 131.8 150.5 150.3 146.4 177.3
MX = Methylxanthine. *There were no statistically significant differences (P S 0.05; paired t test).
,
counting a standard solution, v' is the volume of the flow cell, v is the flow rate, Q is the disintegrations per minute, and B is the background disintegrations per minute as determined from an average determination of radioactivity in a nonradioactive sample (total counts per minute from a nonradioactive sample divided by the number of peaks during that blank run). An additional urine sample (100 pl) was analyzed by liquid scintillation counting to verify the recovery calculated by the HPLC radioactivity monitor. Fecal radioactivity was determined by combustion techniques with aliquots of homogenates prepared in distilled water.5 The logarithm of the total serum theobromine conwas plotted against time after theobromine ingestion. Similar graphs were prepared with data for [8-I4C]theobromine to compare results with those obtained from unlabeled theobromine. The t% of the terminal linear phase of each graph was determined by least-squares linear regression. Total AUC (AUC,,,) was calculated as:
(3)
where F = 1, the fraction of the dose absorbed. After.7 days of chocolate consumption equivalent to 6 mgikgiday theobromine, residual theobromine was present in serum at the time of dosing. Therefore, kinetic parameters for this period were calculated with a steady-state model and the following equations: AUC,,,
= AUC,,,
(4)
*
*
AUC,,
= AUC,,
Cs(24) +-
ß
where AUCo,24 is the area calculated by the linear trapezoidal method, Cs(24) is the serum theobromine concentration at 24 hours, and ß is 0.693/t%. Oral serum clearance (Cl) and apparent volume of distribution (aVd) of theobromine were calculated as: c1 =
F . dose AUC,,
(2)
where T is the theobromine dosing interval of 24 hours. AUCO,, was also calculated from the serum radioactivity data (Eq. 1) to verify the accuracy of the kinetic models chosen for analysis of unlabeled theobromine. Because radioactivity measurements are independent of cumulation effects, a steady-state model would not be appropriate. The relative bioavailability of theobromine in chocolate compared with that in an oral solution of theobromine was calculated as: Relative bioavailability where AUC,,,
=
AUC,, AUC,,
(chocolate) (solution)
(7)
(chocolate) was calculated after a sin-
CLIN PHARMACOL THER APRIL 1985
420 Shively et al.
Table III. Urine radioactivity derived from a 6 mg/kg oral [8-'4C]theobromine dose after methylxanthine abstinence or consumption Percentage of recovered radioactivity*
6-AMMU
7-MU
7-MX
3-MX
TBR
Urine recovery (% of dose)
MX Abstinence M. D. S. H. D. I. K. K. H.M. T. S. I. s. D. S. X
8.2 9.9 9.0 11.3 10.4 10.5 11.1 6.2
9.7 11.2 9.2 8.8 12.1 11.5 13.2 8.4
44.6 43.4 42.3 41.4 44.2 41.8 43.0 34.6
23.5 18.6 21.4 21.7 22.5 16.2 21.3 18.1
13.9 16.3 18.1 16.8 10.8 20.1 11.3 32.6
82.3 86.0 69.57 95.2 88.0 93.5 96.1 80.6
9.6
10.5
41.9
20.4
17.5
86.4
MX Consumption M. D. S. H. D. I. K. K. H. M. T. S . I. s. D. S. -
8.4 11.6 8.6 11.4 10.2 9.5 11.1 9.1
9.6 12.4 8.2 9.2 13.4 11.2 12.8 6.3
42.3 41.3 42.5 41.4 44.5 40.6 41.8 32.5
25.3 17.2 24.7 24.4 18.2 16.5 21 .o 21.7
14.3 11.5 15.9 13.6 13.6 22.1 13.2 30.4
84.9 73.17 81.3 87.6 93.3 84.3 70.27 73.9t
10.0
10.4
41.6
21.1
16.8
81.1
Subject
X
-1
6-AMMU = 6-Amino-5~N-methylfomiylaminol-l-methyluil;7-MU = 7-methyluric acid; 7-MX = 7-methylxanthine;3-MX = 3-methylxanthine; TBR = unchanged theobromine; MX = methylxanthine. *All of the recovered urine radioactivity (O to 12 hr) was present as one of the indicated compounds. tLow recoveries resulted kom incomplete urine collection.
gle, oral, 6 mgikg theobromine dose in dark chocolate and AUC,,, (solution) was calculated after a single, oral, 6 mgikg theobromine dose given as theobromine sodium acetate after 14 days of a methylxanthine-free diet. Because other studies' with both oral and intravenous doses of theobromine revealed that bioavailability of an oral theobromine solution closely approximates 1, the relative bioavailability of theobromine can be assessed by calculation of the ratio of the AUC after different oral dosage forms.
RESULTS Mean serum theobromine t%, aVd, and C1 calculated in 12 normal men after 14 days of abstention from all dietary methylxanthines were 10.0 hours, 0.76 Likg, and 0.88 mllminikg (Table I). These values are in close agreement with those in an earlier studyI7 in this laboratory that defined baseline theobromine kinetics and metabolism during a methylxanthine-free diet. After 7 days of chocolate consumption equivalent to 6 mgikgl day, mean theobromine t%, aVd, and C1 were 9.7 hours, 0.74 L/kg, and 0.90 mlirninikg (Table I). Fig. 1 shows serum theobromine concentrations in each sub-
ject after an oral dose of theobromine sodium acetate after 14 days of methylxanthine abstinence and again after 7 days of chocolate consumption. Serum theobromine concentrations are higher after 7 days of chocolate consumption than they are after abstinence from all methylxanthines. This difference arises from theobromine remaining in serum after 7 days of chocolatL _i consumption. With a steady-state model to calculate AUC, aVd, and C1, these kinetic values can be appropriately measured after 7 days of chocolate consumption. Serum radioactivity data yielded theobromine t% values identical to those obtained by HPLC analysis during both periods of the study (Table II). Calculation of the AUC from radioactivity data according to Eq. 1 (Table II) also indicated kinetic values similar to those in Table I. These results, derived from both labeled [814C] and unlabeled theobromine data, indicated that 7 days of chocolate consumption did not alter theobromine kinetics after 14 days of a methylxanthine-free diet. The cumulative urinary excretion of radioactivity by eight subjects after each received an oral tracer dose (60 FCi) of [8-'4C]the~bromineis listed in Table III.
VOLUME 37 NUMBER 4
Methylxanthine efects on theobromine metabolism 42 1
Table IV. Relative bioavailability of theobromine in chocolate compared with that after an oral solution
Subject
M.D. S. H.
D.S.
Dosage form
t% (hr)
Solution Chocolate Solution Chocolate
9.0 8.0 8.2 7.2 13.3 11.8
Solution
Chocolate ,C
C,, (pglml)
8.8 6.3 10.8
6.9 7.0 6.1
t,", (hr)
0.5 4.3 0.6 2.1 1.0 2.0
P (hr-')
0.077 0.087 0.085
0.097 0.052 0.059
AUC,., (mglL * hr)
aVd (Llkg)
c1 (mllminlkg)
Relative bioavailability
118.6 93.7 108.6 135.6
0.66 0.74 0.65 0.73 0.85
115.1
0.88
0.84 I .O7 0.92 1.18 0.74 0.87
1 .o00 0.790 1.o00 0.781 1 .o00 0.849
84.8
= Maximum concentration; tmax= time to reach Cmax.
After 14 days of methylxanthine abstention, an average of 86.4% of the dose of [8-14C]theobrominewas re,covered in urine. The major metabolite was 7-meth)xanthine, which accountedfor41.9% of the recovered -radioactivity. Other metabolites were 3-methylxanthine, 20.4%; 7-methyluric acid, 10.5%; and 6-amino5[N-methylformylamino]- 1-methyluracil, 9.6%. Unchanged theobromine accounted for 17.5% of the recovered dose. After 7 days of consumption of chocolate containing 6 mglkgiday theobromine, 81.1% of the dose of theobromine was recovered in urine (Table II). Essentially identical amounts of each metabolite and of unchanged theobromine were present in the urine of each subject after chocolate consumption and during abstinence; thus there was no shift in the metabolic profile after subacute consumption of theobromine from a dietary source. Urine samples collected on days 4 and 11 of the 14-day methylxanthine abstinence period indicated that the subjects were compliant and avoided all methylxanthines. While subjects S . H. and R. M . had higher than background amounts of theobromine on day 4, the levels had returned to less than background Jounts by day 11. Fecal elimination of [8-'4C]theobromine4erived radioactivity was determined in two subjects (M. D. and D. S.) to evaluate the extent of elimination of theobromine by this route. Only minor amounts of theobromine were excreted in feces after either methylxanthine abstention or consumption (M. D.: 0.56% and 0.15%; D. S.: 0.54% and 1.42%). These results indicate that most of the theobromine was absorbed in the gastrointestinal tract and that consumption of theobromine as chocolate for 7 days did not alter the fraction of the total radioactivity that was not absorbed after an oral solution. An additional experiment was conducted in three subjects (M. D., S. H., and D. s.)to determine the relative bioavailability of theobromine (6 mg/kg) taken as chocolate and of the same dose taken as an oral solution of
theobromine sodium acetate. If autoinhibition of theobromine elimination exhibited a dose-response relationship that shifted markedly at these particular concentrations of theobromine, a potential difference in theobromine bioavailability between these two dosage forns could explain the differences between our observations and those of Drouillard et Data in Table IV show that the relative bioavailability of theobromine in chocolate is only 78.1% to 84.9% that of an oral solution of theobromine sodium acetate. The lower maximum concentration and the longer time to reach it of theobromine as chocolate indicate a slower rate and extent of theobromine absorption than after dosing from an oral solution (Fig. 2). The clinical chemistry profiles determined in the zero-hour fasting blood specimens of all subjects are listed in Table V. Although several parameters (phosphorus, alanine aminotransferase, protein, and albumin) differed statistically after chocolate consumption according to a paired t test, all values were well within the normal clinical range for adult men.
DISCUSSION In sufficient concentration, theobromine can exert diverse pharmacologic effects such as diuresis, stimulation of the cardiovascular system, relaxation of smooth muscles, and augmentation of glandular secretions. Although not given as a drug, theobromine is widely taken in the form of chocolate and cocoa. Because of potential effects from dietary methylxanthines, factors that influence their systemic concentrations, such as bioavailability from foods that contain them or their capacity to inhibit their own hepatic biotransformation, are of potential clinical interest. This study represents the first investigation of the direct effects of theobromine from a dietary source (chocolate) on its own metabolism and elimination under dietary control of the total number of daily calories (2500) and of the proportion of carbohydrate (45%),
GLIN I'HAKMAWL I H h K
422 Shively e t al.
APRIL 1985
L
10
MD
5
1
0.5
I'
o Theobromine in Solution 8 Theobromine in Dark Chocolate
t 0.1
10
5
1
0.5
o. 1
2o 10
i
DS
5
1
0.5
0.1'
o
4
a
12
16
20
24
Hours After Theobromine Administration Fig. 2. Theobromine serum concentration-time curves after a 6 mg/kg oral dose of theobromine in dark chocolate and in an oral solution as theobromine sodium acetate.
protein (15%), and fat (40%) in those calones. Rigorous control of these nutritional variables is now recognized as necessary to investigate adequately certain kinetic and dynamic effects of dietary manipulation, 24,6~19 Other studies that addressed autoinhibition of methylxanthine metabolism used nondietary dosage forms such as oral suspensions of theobromine,8 gelatin capsules containing theobromine,14 or caffeine and theophylline tablets. l5 Moreover, except for restriction of methylxanthines, subjects in those investigations were under conditions of uncontrolled diet selection. Because methylxanthines are common in foods and beverages such as coffee, tea, cola drinks, or chocolate products, it is important to determine their potential kinetic consequences as a component of the diet. The influence of diet on drug disposition has been well documented. 24.6.19 To determine the metabolic and kinetic effects of theobromine ingestion, we used a dark chocolate that contains more theobromine (0.480 gm per 100gm chocolate) than milk chocolate (O. 153 gm per 100 gm chocolate). *O Subacute administration of theobromine in chocolate for 7 consecutive days in a dose equivalent to 6 mgíkg theobromine had no effect on theobromine elimination or metabolism (Tables I, Ii, and III; Fig. 1). Initially, these results might seem to conflict with those of an earlier study in which the same dose of theobromine in an oral suspension inhibited its own clearance.' It is well e s t a b l i ~ h e d , ~ ~ , "h*owever, ' ~ , ' ~ that factors such as inert ingredients, dosage form, and other formulation variables can markedly influence bioavailability and the pattern of time release. Accordingly, although we gave for 7 days in the form of chocolate the same oral dose of theobromine (6 mg/kg) as previously given' in a suspension of theobromine, our mean relative bioavailability value for the theobromine in chocolate of 80% (Table IV) indicates that the amount of theobromine reaching general circulation is about one-fifth less when taken in chocolate than in an oral solution. As indicated in Table IV and Fig. 2, the rate and extent of absorption of theobromine from chocolate were lower than that of an oral solution. Thus from the point of view of future studies designed to identify interactions between nutritional factors and drugs, the metabolic and therapeutic consequences of a particular chemical can differ markedly when that chemical is taken in the pure compared with the dietary form. Studies of methylxanthines and cocoa by Czok7 reported lower biologic effectiveness of methylxanthines in cocoa products than as pure alkaloids. This difference in effect was attributed to impaired gastrointestinal absorption of methylxanthines because of polyhydroxy-
l ,/
VOLUME 37 NUMBER 4
Methylxanthine efects on theobromine metabolism 423
Table V. Clinical chemistry parameters after 14 days of abstinence from methylxanthines and after 7 days of chocolate consumption equivalent to 6 mg/kg theobromine a day
Subject
TriAlbuCholglycPotasPhosSodium sium Calcium phorus SGOT SGPT BUN Protein min esterol erides Glucose (mEqlL) (mEqlL) (mgldl) (mgidl) (IUIL) (IUIL) (mgldl) (gmldl) (gmidl) (mgidl) (mgldl) (mgldl)
MX Abstinence M. D. 146.9 S. H. 142.9 145.4 D. I. 144.0 K. K. 140.2 T.K. 144.4 M. M. 139.5 H. M. R. M. 143.7 T. S. 147.4
.2;.:. -
D. S . -
X
2 SD
143.4 145.2 141.8
4.55 4.13 4.61 4.71 4.74 4.42 4.46 4.81 3.88 4.40 4.25 4.74
10.1 9.3 9.8 10.5 9.9 9.8 9.7 10.2 9.3 9.3 10.0 8.7
3.3 3.1 3.5 2.9 2.2 2.6 2.9 3.3 3.8 3.5 2.5 2.8
21 19 15 21 13 11 18 11 15 16 28 18
21 7 12 9 6 9 14 6 11 8 13 15
14 10 19 15 13 20 15 17 11 19 17 14
9.0 6.6 8.3 9.0 7.7 8.3 7.6 9.2 8.7 8.2 8.6 7.3
4.9 3.9 4.4 5.3 4.5 4.8 4.6 5.6 5.1 5.2 5.4 4.7
20 1 150 177 208 114 121 146 188 216 184 190 170
65 42 59 99 78 81 75 93 224 47 120 134
85 67 74 76 63 77 65 92 82 84 85 83
143.7 22.4
4.48 20.28
9.7 20.5
3.0 20.5
17
11 24
15 +3
8.2 20.8
4.9 20.5
172 t 33
93
25
2 50
78 29
142.3 146.4 144.9 143.1 146.7 143.1 146.4 146.4 142.4 145.5 142.1
4.59 4.00 4.26 5.01 5.24 4.25 4.50 4.76 4.38 4.65 4.15 4.35
9.9 9.3 9.6 10.3 10.1 9.8 9.9 9.8 9.5 9.3 9.9 8.9
3.8 3.2 3.4 2.8 3.4 3.5 3.1 3.2 4.3 3.7 2.8 2.8
35 16 19 59 14 13 21 9 14 16 21 19
27 10 20 23 8 14 17 5 7 13 15 16
12 11 14 14 12 17 13 9 15 14 16 15
8.2 6.4 8.1 8.9 7.5 8.0 7.0 9.0 8.5 7.8 8.8 6.8
4.4 3.6 4.5 5.3 4.6 4.8 4.3 5.6 4.9 5.0 5.4 4.4
198 153 152 202 i 19 135 137 i 67 225 183 193 147
50 37 32 68 106 99 86 69 305 57 20 1 128
82 73 74 80 72 95 97 83 78 80 92 79
144.6 21.9
4.51 20.36
15* 27
13 22
7.9* 20.9
4.7* 20.6
+ 32
103
82 I 8
8-25
6.6-8.6
4.2-5.4
135-307
MX Consumption M. D. 146.4 S . H. D. I.
K. K. T.K. M. M. H. M. R. M . . T.S. R. S. I. s. D. S . -
X
+I SD
i
Normal )range
.
138-148 3.6-5.0
9.7 20.4
3.3* 20.5
21 213
7.7-10.1
2.2-4.0
3-34
4-35
168
2 79
11-195
71-114
2
MX = Methylxanthine. *Significantiy different (P S 0.05; paired t test) from values after abstinence.
phenols in cocoa or the large fat content. Czok7 demonstrated that caffeine absorption in rats was reduced by approximately 50% when the dose was given 2 hours after a small quantity of olive oil. Thus differences in metabolic response to theobromine in our study compared with data of Drouillard et al.' may arise from reduced theobromine concentrations reaching the liver because of diminished gastrointestinal absorption of theobromine from chocolate. Our observations are in agreement with those of Miners et al., l4 who also found no reduction in theobromine clearance at doses of 200 mg every 8 hours for 4 days. Our results indicate that consumption of large
amounts of chocolate equivalent to 6 mg/kg theobromine a day (approximately two to three dark chocolate bars, 1.45 oz each) did not alter the elimination kinetics or metabolic pattern of theobromine. Since these doses of theobromine are equivalent to three times the usual human consumption level (as determined in marketing research surveys), it may be concluded that the maximum level of theobromine consumption in the form of chocolate in the American diet does not alter its own rate of elimination. We are grateful to David M. White and Joan L. Apgar of Hershey Foods Corporation for their technical assistance.
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CLIN PHARMACOL THER APRIL 1985
release theophylline products. Clin Pharmacokinet 9:95135, 1984. 11. Jones DT, Sears MR: Serum theophylline levels in adult asthmatics: Comparison of oral sustained-release and microcrystalline preparations. NZMed J 92: 196-198,1980. 12. Kappas A, Anderson KE, Conney AH, Alvares AP: Influence of dietary protein and carbohydrate on antipyrine and theophylline metabolism in man. CLINPHARMACOL THER20:643-653, 1976. 13. Lindenbaum J, Mellow MH, Blackstone MO, Butler VP: Variation in biologic availability of digoxin from four preparations. N Eng1 J Med 2851344-1347, 1971. 14. Miners JO, Attwood J, Birkett DJ: Theobromine metabolism in man. Drug Metab Dispos 10:672-675, 1982. 15. Monks TJ, Caldwell J, Smith RL: Influence of methylxanthine-containing foods on theophylline metabolism and kinetics. CLINPHARMACOL THER26:513-524,1979. 16. Monks TJ, Smith RL, Caldwell J: A metabolic and phar macokinetic comparison of theophylline and aminophylline (theophylline ethylenediamine). J Pharm Pharmacol 33:93-97, 1981. 17. Tarka SM, Amaud MJ, Dvorchik BH, Vesell ES: Theobromine kinetics and metabolic disposition. CLINPHARMACOL THER34546-555, 1983. 18. Thompson PJ, Skypala I, Dawson S, McAllister WAC, Turner Warwick M: The effect of diet upon s e m concentrations of theophylline. Br J Clin Pharmacol 16:267270, 1983. 19. Vesell ES: Complex effects of diet on drug disposition. CLINPHARMACOL THER36:285-296, 1984. 20. Zoumas BL, Kreiser WR,Martin RA: Theobromine and caffeine content of chocolate products. J Food Sci 45314-316. 1980.
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