- Email: [email protected]
The Influence of Orally and Parenterally Administered Salts of Terephthalic Acid on Oxytetracycline Serum Levels in Chickens
DIETARY METHIONINE AND SULPHUR AMINO ACIDS IN MEAT REFERENCES Fry, J. L., and W. J. Stadelman, 1960a. Optimum conditions of hydrolysis for microbiological assay of amino acids in poultry meat. 1. Methionine and cystine. J. Ag. Food Chem. (in press). Fry, J. L., and W. J. Stadelman, 1960b. The effect
617
of cooking and carcass part on the methionine and cystine content of poultry meat. Food Research (in press). Ward, W. H., G. H. Binkley and N. S. Snell, 1955. Amino acid composition of normal wool, wool fractions, mohair, feather and feather fractions. Textile Research J. 25: 314-325.
K. E. PRICE, 1 Z. ZOLLI, JR. AND G. A. DONOVAN Chas. Pfizer &" Co., Inc., Terre Haute, Ind. (Received for publication August 28, 1959)
I
T HAS been demonstrated previously that concurrent supplementation of poultry rations with terephthalic acid and oxytetracycline2 results in higher antibiotic serum levels than those obtained with oxytetracycline alone (Price et ah, 1959b; Price and Zolli, 1959a). A possible mode of action by which terephthalic acid potentiates the tetracycline antibiotics has been the subject of a separate report (Price and Zolli, 1959b). In this, it was shown that the relatively soluble sodium salt of terephthalic acid increased oxytetracycline serum levels, whether administered via the gastrointestinal tract or parenterally. Gastrointestinal tract administration was accomplished by injection into a ligated segment of the small intestine, while parenteral administration was by various routes. Although the latter studies were limited, it appeared that the antibiotic serum level response was inversely proportional to the rate of release of the terephthalic 1
Present address: Bristol Laboratories, Syracuse, N.Y. 2 Terramycin, trademark of Chas. Pfizer & Co., Inc.
acid salt from the site of injection, as less potentiation was obtained from administration of terephthalic acid intravenously than intramuscularly. The greatest potentiating effect was with subcutaneous administration. The purpose of the present investigation was to determine the influence of several terephthalic acid salts and the dimethyl ester of terephthalic acid on oxytetracycline serum levels when the antibiotic and these substances were concurrently administered in the feed, in the drinking water, or parenterally. Efforts were made also to establish optimum concentrations of the terephthalate derivatives for maximum potentiation of oxytetracycline. EXPERIMENTAL PROCEDURE Experiment 1. Eighty-five five-weekold male Nichols White Cross chicks were allotted into 17 groups of 5 birds each, groups being approximately equal in weight. Birds were placed in finishing batteries and given the appropriate supplement in a diet which was basically corn-soy and fairly typical of commercial rations. Calcium and phosphorus, which
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
The Influence of Orally and Parenterally Administered Salts of Terephthalic Acid on Oxytetracycline Serum Levels in Chickens
618
K. E. PRICE, Z. ZOIXI, JR. AND G. A. DONOVAN
In one series, rations containing OTC as TM-10, at concentrations of 200 and 500 gm. per ton, were further supplemented with disodium or bis-diethanolamine terephthalate at levels equivalent to 0.1, 0.2 or 0.4% TA. Control rations containing the highest level of TA salts or the antibiotic only were also included. In another series, birds on rations similarly supplemented with antibiotic were concurrently given in the water disodium terephthalate or bis-diethanolamine terephthalate in dilutions to provide the equivalent of 0.05, 0.1, 0.15 or 0.2% free terephthalic acid. Freshly medicated water was made available to the birds daily. Control groups receiving the highest level of the disodium or bis-diethanolamine salts were maintained on an antibioticfree diet. The remaining groups in this experiment were simultaneously medicated through the drinking water with both OTC (at 110 or 275 mg./l. as TPFLC) and disodium or bis-diethanolamine terephthalate. The salts were added so as to provide final concentrations equivalent to 0.05% or 0.1% of the free acid. Water was freshly medicated daily. Control groups receiving the highest level of TA salts or OTC only were included. Group weight gains and feed consumption were determined at the end of 7 days, while water consumption was measured daily. After the birds were weighed, they were heart-bled and serum specimens obtained for antibiotic assay. Assays were conducted as in experiment 1. Experiment 3. One hundred eleven 6week-old straight-run Nichols White Cross birds averaging 1.5 lb. in weight, were placed on the same diet as in experiment 1, both feed and water being offered ad libitum. The birds were randomly allotted into 13 groups of 7 each and two of 10 each. The latter groups were in-
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
have been shown to influence absorption of oxytetracycline from the intestine (Price el al., 1959a), were respectively 1.24% and 0.73% of the ration. Oxytetracycline (OTC) as the commercial product, TM-10, was incorporated in the feed at levels of 200 or 500 gm. per ton. Terephthalic acid (TA), the dimethyl ester of TA, or disodium terephthalate was added to the rations at levels calculated to provide a concentration of 0.375%, calculated as equivalent of free acid. Administration of OTC through drinking water was accomplished by adding the appropriate quantity of Terramycin Poultry Formula Liquid Concentrate (TPFLC) to freshly filled waterers. Antibiotic concentrations in water were 100 and 250 mg./l. Birds receiving each level of OTC were simultaneously given, via the drinking water, a sufficient quantity of a 10% aqueous solution of disodium, bisdiethanolamine, or bis-triethanolamine terephthalate to provide a final TA concentration of 0.1875% in the water. After six days, during which feed and water were given ad libitum, weight gains and feed consumption were measured and blood samples obtained from all birds by heart puncture. Sera were harvested on the day of bleeding and stored at — 60°C. until assayed for antibiotic content by the cylinder-plate method, in which Bacillus cereus var. mycoides (ATCC No. 9634) is employed as the test organism (Grove and Randall, 1955). OTC hydrochloride was used as the standard of comparison. The minimum assayable level of OTC was 0.15 mcg./ml. Experiment 2. A total of 240—4J-weekold straight-run Nichols White Cross chicks were assigned to 48 pens comprising 6 finishing batteries. Five birds were randomly allotted to each pen. The basal diet was similar to that of experiment 1.
619
TEREPHTHALIC ACID AND OXYTETRACYCLINE
Blood samples were taken from wing veins at 3, 6, 24 and 48 hours post injection. Sera were harvested and assayed as described for experiment 1. RESULTS
The first experiment was conducted to determine the relative ability of terephthalic acid, its dimethyl ester and its disodium, bis-triethanolamine and bismonoethanolamine salts to enhance oxytetracycline serum levels. Table 1 shows serum OTC levels when both antibiotic and the acid or one of its derivatives were administered simultaneously in feed. TABLE 1.—Effect of terephthalic acid and its derivatives administered in feed concurrently •with oxytetracycline on antibiotic Hood serum levels in chickens
Treatment
Av. 6-day O T C serum Percent inlevels (5 crease over control birds) mcg./ml.
OTC, 200 g m . / t o n <0.1502 0.260 -fTerephthalic acid (0.375%) 0.301 + D i s o d i u m terephthalate (0.375%)' 0.217 + D i m e t h y l terephthalate (0.375%)' OTC, 500 g m . / t o n -(-Terephthalic acid (0.375%) +Disodium terephthalate (0.375%)' + D i m e t h y l terephthalate (0.375%)> Non-medicated control 1
> 71.0 > 98.0 > 43.0
0.355 0.686 0.715 0.538
93.0 101.0 51.0
<0.150
—
Added at a concentration equivalent to 0.375% of the free acid.2 Two of five samples gave detectable levels.
TABLE 2.—Effect of various terephthalic acid derivatives administered concurrently in drinking water with oxytetracycline on antibiotic blood serum levels in chickens
Treatment
Av. 6-day OTC serum Percent increase over levels control (5 birds) mcg./ml.
<0.15 OTC, 100 mg./liter -(-disodium terephthalate (0.1875%) < 0 . 1 5 2 -j-monoethanolamine terephthalate (0.1875%)' <0.15= -f-triethanolamine terephthalate (0.1875%)' 0.267 OTC, 250 mg./liter + d i s o d i u m terephthalate (0.1875%) -j-monoethanolamine terephthalate (0.1875%)' -f-triethanolamine terephthalate (0.1875%)' Non-medicated control
—. — — > 79.0
—
0.275 0.538
96.0
0.582
112.0
0.535
95.0
<0.15
—
' Added at a concentration equivalent to 0.1875% of the free acid. 2 Two of 5 serum samples gave detectable levels.
It can be observed that all additives increased OTC serum levels over controls. However, at both concentrations of antibiotic, the free acid and the sodium salt exerted a greater potentiating effect than the dimethyl ester. The ability of the disodium salt to increase serum OTC levels seemed slightly greater than that of the acid. It is of interest that the 0.375% concentration of these two additives potentiated both the 200 gm./ton and the 500 gm./ton levels of OTC to about the same degree. Table 2 shows serum OTC levels when both antibiotic and soluble terephthalates were simultaneously administered in water. At 100 mg. OTC/liter, potentiation of OTC serum levels was obtained only with the triethanolamine salt. However, there were indications that the other salts influenced OTC serum levels, since, with each, 2 of 5 serum samples had antibiotic levels in excess of 0.15 mcg./ml. in contrast to the control for which none of the samples showed detectable levels. When OTC was administered in the drinking water at 250 mg./liter, all three salts exhibited similar potentiating ability.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
jected subcutaneously with 25 mg. or 50 mg. doses of OTC, whereas the others were injected at the same subcutaneous site with freshly prepared solutions containing both antibiotic and disodium terephthalate. Doses were prepared by adding graded concentrations of disodium terephthalate in 2.0 ml. of aqueous solution to 1 or 2 ml. of Terramycin Injectable Solution (TIS), which contains 25 mg. OTC per ml. Final concentrations of disodium terephthalate were 25, 50, 75, 100, 150 or 200 mg. per dose. Control groups receiving saline, antibiotic, or the highest level of disodium terephthalate only were maintained.
620
K. E. PRICE, Z. ZOLLI, J R . AND G. A. DONOVAN
Control
ble ethanolamine salt, bis-diethanolamine, required to give the maximum potentiating effect with feed- and wateradministered OTC. Figure 1 shows the results obtained with graded levels of these salts in conjunction with OTC added to feed at 200 gm./ton, or to drinking water at 110 mg./l. When OTC and TA salts were administered in the feed, both the bis-diethanolamine and disodium salts of TA increased antibiotic serum levels over the control. Although the highest antibiotic serum levels occurred with 0.4% of both salts, serum levels at the 0.2% concentration were only slightly lower. Similar potentiation of serum levels from feed-administered antibiotic was obtained when graded concentrations of the salts were given in the drinking water. However, the maximum increase was obtained with salt concentrations of
Bis-diethanolamine terephthalate
F ^
OTC+TA SALTS IN . FEED
OTC IN FEED; TA SALTS IN WATER
OO 01 0-2 0-4
0 0 0 0 5 01 0|5 0 2
%TASalt* * Free terephthalfc acid equivalent
%TASalt*
Disodium terephthalate
OTC + TA SALTS IN WATER
0.05 0 1 0-2 %TASalt*
FIG. 1. The influence of various concentrations of bis-diethanolamine or disodium salts of terephthalic acid (TA) upon serum levels of oxytetracycline (OTC) administered at 200 gm. per ton in feed or HOppm. in drinking water.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
Antibiotic serum levels produced in the presence of the salts were approximately 100% greater than that of the control. In general, OTC blood serum levels were somewhat higher for birds receiving antibiotic in the feed than in water. This may be explained by relative quantities of feed and water consumed. During the six-day test period, each bird consumed an average of 0.92 pound of feed and 1.42 pounds of water. The resulting water-tofeed ratio (1.65:1) was considerably lower than the 2:1 ratio assumed in attempting to set feed and water antibiotic levels so as to provide equal intakes from each source. None of the treatments exerted any influence on weight gains or feed or water consumption. Experiment 2 was conducted to determine the minimum concentrations of disodium terephthalate and a more solu-
621
TEREPHTHALIC ACID AND OXYTETRACYCLINE Control
£8583
Bis-dlethanolamine terephthalate
20
OTC IN FEED; TA SALTS IN WATER
0 0 01 0-2 0-4
0 0 0 0 5 01 015 0-2
r-
OTC + TA SALTS IN WATER
1-6
f
1-2
0:8
O
0-4
%TASalt* * Free terephthalic acid equivalent
%TASalt*
0 0 0 0 5 01 %TASalt*
FIG. 2. The influence of various concentrations of bis-diethanolamine or disodium salts of terephthalic acid (TA) upon serum levels of oxytetracycline (OTC) administered at 500 gm. per ton of feed or 275 ppm. in drinking water.
0.05%, considerably less than the level required for comparable potentiation by feed-administered salts. W hen OTC and TA derivatives were both administered in water, serum antibiotic levels were higher than those obtained when antibiotic was administered in the feed. This can be explained by the fact that antibiotic intake in water in this experiment was approximately 25% higher than via feed. Maximum potentiation of water-administered antibiotic occurred at the 0.05% level of both the bis-diethanolamine and disodium salts. Figure 2 shows the average antibiotic blood serum levels obtained in the presence of graded levels of terephthalates when OTC was administered in the feed at 500 gm./ton or in the drinking water at 275 mg./l. It can be observed that
antibiotic serum concentrations at these levels of supplementation were at least twice as high as those obtained at lower OTC feed, or water levels (Figure 1). Maximum potentiation with the 500 gm./ ton level of OTC was obtained with 0.2% of feed-administered TA salts. When the salts were added to the drinking water and the antibiotic to the feed, blood serum concentrations up to 3 times as high as the control were observed. In contrast to the response produced at the lower feed level of OTC (200 gm./ton), where maximum potentiation occurred at 0.05% of TA salts, the higher TA salt levels with 500 gm. OTC/ton induced progressively greater antibiotic serum concentrations without clear indication of a maximum. Simultaneous administration in water
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
0TC«TA SALTS IN FEED
Disodium terephthalate
622
K. E. PRICE, Z. ZOLLI, JR. AND G. A. DONOVAN
3 HOURS POST INJECTION
the bis-diethanolamine or disodium salts of TA were observed in this 7-day trial. The influence of disodium terephthalate injected concurrently with OTC by the subcutaneous route on antibiotic blood serum levels was determined in the third experiment. Serum levels obtained from 3.0 ml. of solution containing 25 mg. of OTC alone or with graded quantities of disodium terephthalate are shown in Figure 3. Although, at 3 hours, serum samples from birds receiving all levels of disodium terephthalate contained higher average antibiotic concentrations than the control, responses were erratic and not large in most instances. Samples taken at 6 hours post injection, however, showed a consistent increase in antibiotic concentration as the level of disodium terephthalate was raised. The maximum blood level, which was obtained with 150 mg. of the salt, exceeded the control by At 24 hours post injection, responses
6 HOURS POST INJECTION
IOO Control
m
80 Disodium terephthalate
60-
•
40
2 4 HOURS POST INJECTION
20
O
O »
N
O
Q
O
m t- o •> o
O
IO O U> O O O Ol K) t» O JO Q
-r-rTT
O
Dose of disodium terephthalate-mg.
FIG. 3. The influence of disodium terephthalate given by subcutaneous injection concurrently with 25 mg. oxytetracycline upon antibiotic blood serum concentration.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
of both antibiotic (275 mg./l.) and TA salts also resulted in OTC serum level increases somewhat larger than those obtained with antibiotic and salts in the feed. A maximum response was suggested at 0.05% to 0.1% of TA salt. In this experiment, the ratio of water consumption to feed consumption was greater than 2:1; hence, intake of TA salts was greater by the groups receiving salts in the water than by those receiving salts in the feed. This difference may account for the relatively lower antibiotic serum levels obtained when both OTC and TA salts were feed administered. Comparison of the data shown in Figures 1 and 2 indicates that the degree of potentiation was greater with the higher level of antibiotic than with the lower, even though the range of TA salt levels was the same in the two series. No marked or consistent differences in weight gains or feed or water consumption due to administration of any level of
623
T E R E P H T H A L I C A C I D AND OXYTETRACYCLINE 3 HOURS POST INJECTION
zoo
6 HOURS POST INJECTION Control
Hi
16 0 Disodium i—i terephthalate '—'
"
80
-
r^ •
24 HOURS POST INJECTION
40
•
nfTTlTh
,•;., O
IO O IO O
N
O
O
O in O u> O O O M
es O no —
~"
CVJ
in N
O IO O — — CM
O m O m o o O N I) S O «l O
— —
Dose of disodium terephthalate-mg.
FIG. 4. The influence of disodium terephthalate given by subcutaneous injection concurrently with SO mg. oxytetracycline upon antibiotic blood serum concentration in chickens. still occurred to the higher levels of disodium terephthalate. Here, average serum concentrations were approximately 0.5 meg./ml. for the 150 and 200 mg. doses, as compared to 0.25 mcg./ml. for the control. No d a t a are exhibited for samples taken at 48 hours, since the majority did not show detectable antibiotic serum concentrations. Results obtained when O T C was injected at a higher dosage level are shown in Figure 4. Fifty 1 mg. antibiotic doses were given subcutaneously in 4.0 ml. total volume of solution, which also contained graded levels of disodium terephthalate. Antibiotic serum concentration in the control (non-potentiated) group at 3 hours was 12.3 mcg./ml. from 50 mg. antibiotic, as compared to 8.1 mcg./ml. obtained from the 25 mg. dose (Figure 3). I n contrast to the erratic 3-hour results obtained with the 25 mg. dose of antibiotic, all birds receiving 50 mg. antibiotic concurrently with disodium tereph-
thalate had markedly higher OTC serum concentrations a t 3 hours than did the control. Doses of the salt as low as 25 mg. per bird gave increases as great or greater t h a n 150 mg. and 200 mg. doses. By 6 hours post injection, the antibiotic serum concentration of the control had dropped to 8.7 mcg./ml., while concentrations in the groups injected with disodium terephthalate were maintained a t levels ranging from 12 to 16 mcg./ml. No relationship of potentiation response to T A salt dose was suggested. Moderate b u t consistent increases in serum antibiotic levels due to injection of disodium terephthalate were still apparent a t 24 hours post injection. Again, no effect of T A salt dosage level was apparent. At 48 hours post injection, m a n y samples did not show detectable O T C serum concentrations. D a t a for this time interval therefore are not given. The degree of potentiation at 6 hours somewhat exceeded t h a t obtained at 3
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
120
624
K. E. PRICE, Z. ZOLLI, J R . AND G. A. DONOVAN
and Zolli, 1959b). Although no explanation for this apparent anomaly is available at present, it may be that some property of the ester, such as its absorption or excretion rate, may account for the DISCUSSION observed variability in potentiating acPotentiation of oxytetracycline was tivity. In rats, preliminary findings have accomplished by oral and parenteral suggested that there are marked differadministration of various salts of tereph- ences between terephthalic acid and its thalic acid. Although data were too limited dimethyl ester with respect to the quanto be conclusive, there was an indication tity of the drug that can be found in the that solubility of a terephthalic compound urine following oral administration (Von may not be an important factor in its Fossen, 1959). Data on the excretion rate relative potentiating ability. Despite the of dimethyl terephthalate in chickens are extreme differences in the solubilities of not yet available. Limited experiments terephthalic acid and its derivatives have indicated that 15% to 25% of an (Table 3), activity of the acid was not orally administered dose of terephthalic shown to be appreciably less than that of acid can be recovered in the urine of the its much more soluble salts. Dimethyl- chicken (Price et al., unpublished data). terephthalate, which like TA, has only The results presented in this report slight solubility in water, had somewhat show that, by the use of soluble salts of less potentiating ability than the acid or terephthalic acid, oxytetracycline can be its salts. potentiated through the drinking water Despite the fact that the dimethyl as well as through the feed. In addition, ester of terephthalic acid was the least the data show that simultaneous paractive of all compounds tested, a sub- enteral administration of disodium terephstantial increase in OTC serum level was thalate and oxytetracycline provides achieved when this compound was fed higher antibiotic serum levels than concontinuously. This is of special interest, trols without a potentiating agent for at since it was previously found that simul- least twenty-four hours post injection. taneous injection of the ester with OTC Although data obtained were not preinto the ligated duodenal loop of chickens cise enough to define optimum levels of did not appreciably increase antibiotic the terephthalic acid derivatives, on the serum levels, whereas the acid did (Price average it appeared that near-maximum increases in antibiotic serum levels were TABLE 3.—Solubilities of terephthalic obtained with 0.05% to 0.15% concenacid and derivatives trations of the salts in the drinking water. Solubility in These concentrations are about | to f Compound water at 20°C, that required to produce near-maximum gm./lOO ml. 1 antibiotic serum level increases when adTerephthalic acid 0.0016 ministration of the salts was through the Dimethyl terephthalate <0.0016 Disodium terephthalate 7-10 feed. However, this would not necessarily Bis-monoethanolamine terephthalate 20-25 indicate greater efficacy of water-adminBis-triethanolamine terephthalate 20-25 Bis-diethanolamine terephthalate 30-35 istered salts, since the greater consump1 tion of water as compared to feed would As equivalent of free terephthalic acid.
hours with both levels of antibiotic when serum concentrations for each time interval resulting from all doses of TA salts were pooled.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
TEREPHTHALIC ACID AND OXYTETRACYCLINE
SUMMARY
This study concerned potentiation of serum concentrations of oxytetracycline (OTC) by terephthalic acid (TA), its sodium salt, and several of its soluble ethanolamine salts. Administration via water, feed and subcutaneous routes was investigated for both the antibiotic and the potentiating agents. The antibiotic was given at two levels, and the potentiating agents were administered at graded levels. Results show that TA and its soluble salts given by all three routes increase serum OTC levels of chickens over those of controls which received antibiotic only. When antibiotic was administered orally, degree of potentiation appeared to be greater with 500 gm./ton in feed, or 275 mg./l. in water, than it was with 200 gm./ton or 110 mg./l. This occurred whether TA salts were administered in feed or water. The data obtained did not permit determination of optimum levels of orally administered terephthalates for maximum potentiating activity. I t was indicated, however, that, with the levels -of OTC studied, maximum increases in serum
levels over controls would be obtained with 0.05% to 0.15% equivalent of free acid of soluble TA derivatives in water, and from two to four times this in feed. Because water intake was greater than feed intake, these percentages are equivalent to somewhat similar aggregate intakes by the two routes, and are not indicative of a greater effectiveness by one route as compared with the other. The data were not sufficient for estimating the optimum potentiating level of disodium terephthalate when administered subcutaneously. All doses definitely potentiated OTC serum levels, but those providing near maxima ranged from 25 mg. to 150 mg. per 1.5 pound bird under different conditions. The factor of time was studied with antibiotic and potentiating agents, both administered subcutaneously. I t was found that, on the average, the degree of potentiation at six hours post-injection was somewhat greater than at three hours with the doses used. ACKNOWLEDGMENT
The authors are grateful to Dr. A. P. Collins for the preparation of the terephthalate compounds used in this study and to Dr. D. F. Chichester for assistance in preparation of the manuscript. REFERENCES Grove, D. C , and W. A. Randall, 19SS. Assay Methods of Antibiotics, New York, Medical Encyclopedia, Inc. Price, K. E., Z. Zolli, Jr., J. C. Atkinson, A. P. Collins and H. G. Luther, 1959a. Antibiotic inhibitors. III. Reversal of calcium inhibition of intestinal absorption of oxytetracycline in chickens by certain acids and acid salts. Antibiotics Annual 1958-59, Medical Encyclopeida, Inc., New York, pp. 1020-1032. Price, K. E., Z. Zolli, Jr. and J. H. Hare, 1959b. Effect of dietary calcium-phosphorus adjustment and/or supplementation with terephthalic acid
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
probably result in a nearly comparable intake of TA salts by the two routes. Insufficient data are available for estimating the optimum level of disodium terephthalate required for maximum potentiation when this salt is administered parenterally. In some cases, doses as low as 25 mg. in a 1.5 pound bird were sufficient to yield near-maximum serum levels. In others, doses up to 150 mg. per bird were required for maximum potentiation. Despite this marked variation, there was a clearcut increase in OTC serum levels obtained with all dosages of disodium terephthalate.
625
626
K. E. PRICE, Z. ZOLLI, J R . AND G. A. DONOVAN
upon serum antibiotic levels of chickens. Poultry Sci. 38: 233-237. Price, K. E., and Z. Zolli, Jr., 1959a. The influence of dietary calcium-phosphorus and terephthalic acid on antibiotic control of experimental infectious synovitis. Avian Diseases, 3: 135-156.
Price, K. E., and Z. Zolli, Jr. 1959b. The influence of terephthalic acid on oxytetracycline serum levels in chickens. Studies on mode of action. I. Avian Diseases, 3: 157-169. Van Fossen, P., 1959. Personal communication.
PRAN VOHRA,* B. W. LANGER, JR. AND F. H. KRATZER Department of Poultry Husbandry, University of California, Davis (Received for publication August 31, 1959)
in excess than C14H? of methionine into methylaminoethanol. Young el al. (1955) found that choline requirement of chickens was decreased by dietary folic acid. Also, choline could be replaced by an equimolar quantity of monomethylaminoethanol plus betaine, either in presence or absence of folic acid. Vitamin BJ2 spared choline requirements of poults for optimum growth (Kratzer, 1952) and appeared to be involved in methyl synthesis (Vohra et al. 1956). In vitamin B^-depleted chicks and rats monomethylaminoethanol plus betaine were as effective as equimolar decarboxylation Serine > aminoethanol concentrations of dietary choline and methionine. However, vitamin Bi2 im"active HCHO" proved growth markedly if betaine con> N-hydroxymethylaminocentration was marginal in the ration reduction ethanol > methylaminoethanol (Young e/aZ., 1954,1957). In the present experiments, an attempt "active HCHO" > N-hydroxymethyl-N- has been made to investigate Stekol's hypothesis to throw some light on the reduction methylaminoethanol >• dimethyl- methyl acceptors for the biosynthesis of choline. "active methionine CH 3 " aminoethanol >
F
OR the biosynthesis of choline, Jukes (1947) and Artom (1952) proposed a scheme for the stepwise addition of methyl groups from methionine via transmethylation to aminoethanol. However, Stekol et al. (1955) demonstrated that dimethylaminoethanol was the only direct acceptor of the methyl group of methionine for choline biosynthesis in rats, the other 2 methyl groups were produced by de novo synthesis involving folic acid. Stekol (1958) postulated the following stepwise sequence for choline biosynthesis:
choline. The de novo synthesis of methyl groups was supported by studies of Nye (1956) in a mutant strain of Neurospora crassa where formate-C w was incorporated far * Present address: BOCM Poultry Demonstration Farm, Stoke Mandeville, Bucks, U.K.
EXPERIMENTAL
N-Hydroxymethylserine: The barium salt of N-hydroxymethylserine has been synthesized (Levene and Schormuller, 1934) but during drying of the product, it lost a molecule of water. The sodium salt of Nhydroxymethylserine was prepared essentially the same way. One mole of
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
Methyl Acceptors for the Biosynthesis of Choline
617
of cooking and carcass part on the methionine and cystine content of poultry meat. Food Research (in press). Ward, W. H., G. H. Binkley and N. S. Snell, 1955. Amino acid composition of normal wool, wool fractions, mohair, feather and feather fractions. Textile Research J. 25: 314-325.
K. E. PRICE, 1 Z. ZOLLI, JR. AND G. A. DONOVAN Chas. Pfizer &" Co., Inc., Terre Haute, Ind. (Received for publication August 28, 1959)
I
T HAS been demonstrated previously that concurrent supplementation of poultry rations with terephthalic acid and oxytetracycline2 results in higher antibiotic serum levels than those obtained with oxytetracycline alone (Price et ah, 1959b; Price and Zolli, 1959a). A possible mode of action by which terephthalic acid potentiates the tetracycline antibiotics has been the subject of a separate report (Price and Zolli, 1959b). In this, it was shown that the relatively soluble sodium salt of terephthalic acid increased oxytetracycline serum levels, whether administered via the gastrointestinal tract or parenterally. Gastrointestinal tract administration was accomplished by injection into a ligated segment of the small intestine, while parenteral administration was by various routes. Although the latter studies were limited, it appeared that the antibiotic serum level response was inversely proportional to the rate of release of the terephthalic 1
Present address: Bristol Laboratories, Syracuse, N.Y. 2 Terramycin, trademark of Chas. Pfizer & Co., Inc.
acid salt from the site of injection, as less potentiation was obtained from administration of terephthalic acid intravenously than intramuscularly. The greatest potentiating effect was with subcutaneous administration. The purpose of the present investigation was to determine the influence of several terephthalic acid salts and the dimethyl ester of terephthalic acid on oxytetracycline serum levels when the antibiotic and these substances were concurrently administered in the feed, in the drinking water, or parenterally. Efforts were made also to establish optimum concentrations of the terephthalate derivatives for maximum potentiation of oxytetracycline. EXPERIMENTAL PROCEDURE Experiment 1. Eighty-five five-weekold male Nichols White Cross chicks were allotted into 17 groups of 5 birds each, groups being approximately equal in weight. Birds were placed in finishing batteries and given the appropriate supplement in a diet which was basically corn-soy and fairly typical of commercial rations. Calcium and phosphorus, which
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
The Influence of Orally and Parenterally Administered Salts of Terephthalic Acid on Oxytetracycline Serum Levels in Chickens
618
K. E. PRICE, Z. ZOIXI, JR. AND G. A. DONOVAN
In one series, rations containing OTC as TM-10, at concentrations of 200 and 500 gm. per ton, were further supplemented with disodium or bis-diethanolamine terephthalate at levels equivalent to 0.1, 0.2 or 0.4% TA. Control rations containing the highest level of TA salts or the antibiotic only were also included. In another series, birds on rations similarly supplemented with antibiotic were concurrently given in the water disodium terephthalate or bis-diethanolamine terephthalate in dilutions to provide the equivalent of 0.05, 0.1, 0.15 or 0.2% free terephthalic acid. Freshly medicated water was made available to the birds daily. Control groups receiving the highest level of the disodium or bis-diethanolamine salts were maintained on an antibioticfree diet. The remaining groups in this experiment were simultaneously medicated through the drinking water with both OTC (at 110 or 275 mg./l. as TPFLC) and disodium or bis-diethanolamine terephthalate. The salts were added so as to provide final concentrations equivalent to 0.05% or 0.1% of the free acid. Water was freshly medicated daily. Control groups receiving the highest level of TA salts or OTC only were included. Group weight gains and feed consumption were determined at the end of 7 days, while water consumption was measured daily. After the birds were weighed, they were heart-bled and serum specimens obtained for antibiotic assay. Assays were conducted as in experiment 1. Experiment 3. One hundred eleven 6week-old straight-run Nichols White Cross birds averaging 1.5 lb. in weight, were placed on the same diet as in experiment 1, both feed and water being offered ad libitum. The birds were randomly allotted into 13 groups of 7 each and two of 10 each. The latter groups were in-
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
have been shown to influence absorption of oxytetracycline from the intestine (Price el al., 1959a), were respectively 1.24% and 0.73% of the ration. Oxytetracycline (OTC) as the commercial product, TM-10, was incorporated in the feed at levels of 200 or 500 gm. per ton. Terephthalic acid (TA), the dimethyl ester of TA, or disodium terephthalate was added to the rations at levels calculated to provide a concentration of 0.375%, calculated as equivalent of free acid. Administration of OTC through drinking water was accomplished by adding the appropriate quantity of Terramycin Poultry Formula Liquid Concentrate (TPFLC) to freshly filled waterers. Antibiotic concentrations in water were 100 and 250 mg./l. Birds receiving each level of OTC were simultaneously given, via the drinking water, a sufficient quantity of a 10% aqueous solution of disodium, bisdiethanolamine, or bis-triethanolamine terephthalate to provide a final TA concentration of 0.1875% in the water. After six days, during which feed and water were given ad libitum, weight gains and feed consumption were measured and blood samples obtained from all birds by heart puncture. Sera were harvested on the day of bleeding and stored at — 60°C. until assayed for antibiotic content by the cylinder-plate method, in which Bacillus cereus var. mycoides (ATCC No. 9634) is employed as the test organism (Grove and Randall, 1955). OTC hydrochloride was used as the standard of comparison. The minimum assayable level of OTC was 0.15 mcg./ml. Experiment 2. A total of 240—4J-weekold straight-run Nichols White Cross chicks were assigned to 48 pens comprising 6 finishing batteries. Five birds were randomly allotted to each pen. The basal diet was similar to that of experiment 1.
619
TEREPHTHALIC ACID AND OXYTETRACYCLINE
Blood samples were taken from wing veins at 3, 6, 24 and 48 hours post injection. Sera were harvested and assayed as described for experiment 1. RESULTS
The first experiment was conducted to determine the relative ability of terephthalic acid, its dimethyl ester and its disodium, bis-triethanolamine and bismonoethanolamine salts to enhance oxytetracycline serum levels. Table 1 shows serum OTC levels when both antibiotic and the acid or one of its derivatives were administered simultaneously in feed. TABLE 1.—Effect of terephthalic acid and its derivatives administered in feed concurrently •with oxytetracycline on antibiotic Hood serum levels in chickens
Treatment
Av. 6-day O T C serum Percent inlevels (5 crease over control birds) mcg./ml.
OTC, 200 g m . / t o n <0.1502 0.260 -fTerephthalic acid (0.375%) 0.301 + D i s o d i u m terephthalate (0.375%)' 0.217 + D i m e t h y l terephthalate (0.375%)' OTC, 500 g m . / t o n -(-Terephthalic acid (0.375%) +Disodium terephthalate (0.375%)' + D i m e t h y l terephthalate (0.375%)> Non-medicated control 1
> 71.0 > 98.0 > 43.0
0.355 0.686 0.715 0.538
93.0 101.0 51.0
<0.150
—
Added at a concentration equivalent to 0.375% of the free acid.2 Two of five samples gave detectable levels.
TABLE 2.—Effect of various terephthalic acid derivatives administered concurrently in drinking water with oxytetracycline on antibiotic blood serum levels in chickens
Treatment
Av. 6-day OTC serum Percent increase over levels control (5 birds) mcg./ml.
<0.15 OTC, 100 mg./liter -(-disodium terephthalate (0.1875%) < 0 . 1 5 2 -j-monoethanolamine terephthalate (0.1875%)' <0.15= -f-triethanolamine terephthalate (0.1875%)' 0.267 OTC, 250 mg./liter + d i s o d i u m terephthalate (0.1875%) -j-monoethanolamine terephthalate (0.1875%)' -f-triethanolamine terephthalate (0.1875%)' Non-medicated control
—. — — > 79.0
—
0.275 0.538
96.0
0.582
112.0
0.535
95.0
<0.15
—
' Added at a concentration equivalent to 0.1875% of the free acid. 2 Two of 5 serum samples gave detectable levels.
It can be observed that all additives increased OTC serum levels over controls. However, at both concentrations of antibiotic, the free acid and the sodium salt exerted a greater potentiating effect than the dimethyl ester. The ability of the disodium salt to increase serum OTC levels seemed slightly greater than that of the acid. It is of interest that the 0.375% concentration of these two additives potentiated both the 200 gm./ton and the 500 gm./ton levels of OTC to about the same degree. Table 2 shows serum OTC levels when both antibiotic and soluble terephthalates were simultaneously administered in water. At 100 mg. OTC/liter, potentiation of OTC serum levels was obtained only with the triethanolamine salt. However, there were indications that the other salts influenced OTC serum levels, since, with each, 2 of 5 serum samples had antibiotic levels in excess of 0.15 mcg./ml. in contrast to the control for which none of the samples showed detectable levels. When OTC was administered in the drinking water at 250 mg./liter, all three salts exhibited similar potentiating ability.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
jected subcutaneously with 25 mg. or 50 mg. doses of OTC, whereas the others were injected at the same subcutaneous site with freshly prepared solutions containing both antibiotic and disodium terephthalate. Doses were prepared by adding graded concentrations of disodium terephthalate in 2.0 ml. of aqueous solution to 1 or 2 ml. of Terramycin Injectable Solution (TIS), which contains 25 mg. OTC per ml. Final concentrations of disodium terephthalate were 25, 50, 75, 100, 150 or 200 mg. per dose. Control groups receiving saline, antibiotic, or the highest level of disodium terephthalate only were maintained.
620
K. E. PRICE, Z. ZOLLI, J R . AND G. A. DONOVAN
Control
ble ethanolamine salt, bis-diethanolamine, required to give the maximum potentiating effect with feed- and wateradministered OTC. Figure 1 shows the results obtained with graded levels of these salts in conjunction with OTC added to feed at 200 gm./ton, or to drinking water at 110 mg./l. When OTC and TA salts were administered in the feed, both the bis-diethanolamine and disodium salts of TA increased antibiotic serum levels over the control. Although the highest antibiotic serum levels occurred with 0.4% of both salts, serum levels at the 0.2% concentration were only slightly lower. Similar potentiation of serum levels from feed-administered antibiotic was obtained when graded concentrations of the salts were given in the drinking water. However, the maximum increase was obtained with salt concentrations of
Bis-diethanolamine terephthalate
F ^
OTC+TA SALTS IN . FEED
OTC IN FEED; TA SALTS IN WATER
OO 01 0-2 0-4
0 0 0 0 5 01 0|5 0 2
%TASalt* * Free terephthalfc acid equivalent
%TASalt*
Disodium terephthalate
OTC + TA SALTS IN WATER
0.05 0 1 0-2 %TASalt*
FIG. 1. The influence of various concentrations of bis-diethanolamine or disodium salts of terephthalic acid (TA) upon serum levels of oxytetracycline (OTC) administered at 200 gm. per ton in feed or HOppm. in drinking water.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
Antibiotic serum levels produced in the presence of the salts were approximately 100% greater than that of the control. In general, OTC blood serum levels were somewhat higher for birds receiving antibiotic in the feed than in water. This may be explained by relative quantities of feed and water consumed. During the six-day test period, each bird consumed an average of 0.92 pound of feed and 1.42 pounds of water. The resulting water-tofeed ratio (1.65:1) was considerably lower than the 2:1 ratio assumed in attempting to set feed and water antibiotic levels so as to provide equal intakes from each source. None of the treatments exerted any influence on weight gains or feed or water consumption. Experiment 2 was conducted to determine the minimum concentrations of disodium terephthalate and a more solu-
621
TEREPHTHALIC ACID AND OXYTETRACYCLINE Control
£8583
Bis-dlethanolamine terephthalate
20
OTC IN FEED; TA SALTS IN WATER
0 0 01 0-2 0-4
0 0 0 0 5 01 015 0-2
r-
OTC + TA SALTS IN WATER
1-6
f
1-2
0:8
O
0-4
%TASalt* * Free terephthalic acid equivalent
%TASalt*
0 0 0 0 5 01 %TASalt*
FIG. 2. The influence of various concentrations of bis-diethanolamine or disodium salts of terephthalic acid (TA) upon serum levels of oxytetracycline (OTC) administered at 500 gm. per ton of feed or 275 ppm. in drinking water.
0.05%, considerably less than the level required for comparable potentiation by feed-administered salts. W hen OTC and TA derivatives were both administered in water, serum antibiotic levels were higher than those obtained when antibiotic was administered in the feed. This can be explained by the fact that antibiotic intake in water in this experiment was approximately 25% higher than via feed. Maximum potentiation of water-administered antibiotic occurred at the 0.05% level of both the bis-diethanolamine and disodium salts. Figure 2 shows the average antibiotic blood serum levels obtained in the presence of graded levels of terephthalates when OTC was administered in the feed at 500 gm./ton or in the drinking water at 275 mg./l. It can be observed that
antibiotic serum concentrations at these levels of supplementation were at least twice as high as those obtained at lower OTC feed, or water levels (Figure 1). Maximum potentiation with the 500 gm./ ton level of OTC was obtained with 0.2% of feed-administered TA salts. When the salts were added to the drinking water and the antibiotic to the feed, blood serum concentrations up to 3 times as high as the control were observed. In contrast to the response produced at the lower feed level of OTC (200 gm./ton), where maximum potentiation occurred at 0.05% of TA salts, the higher TA salt levels with 500 gm. OTC/ton induced progressively greater antibiotic serum concentrations without clear indication of a maximum. Simultaneous administration in water
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
0TC«TA SALTS IN FEED
Disodium terephthalate
622
K. E. PRICE, Z. ZOLLI, JR. AND G. A. DONOVAN
3 HOURS POST INJECTION
the bis-diethanolamine or disodium salts of TA were observed in this 7-day trial. The influence of disodium terephthalate injected concurrently with OTC by the subcutaneous route on antibiotic blood serum levels was determined in the third experiment. Serum levels obtained from 3.0 ml. of solution containing 25 mg. of OTC alone or with graded quantities of disodium terephthalate are shown in Figure 3. Although, at 3 hours, serum samples from birds receiving all levels of disodium terephthalate contained higher average antibiotic concentrations than the control, responses were erratic and not large in most instances. Samples taken at 6 hours post injection, however, showed a consistent increase in antibiotic concentration as the level of disodium terephthalate was raised. The maximum blood level, which was obtained with 150 mg. of the salt, exceeded the control by At 24 hours post injection, responses
6 HOURS POST INJECTION
IOO Control
m
80 Disodium terephthalate
60-
•
40
2 4 HOURS POST INJECTION
20
O
N
O
Q
O
m t- o •> o
O
IO O U> O O O Ol K) t» O JO Q
-r-rTT
O
Dose of disodium terephthalate-mg.
FIG. 3. The influence of disodium terephthalate given by subcutaneous injection concurrently with 25 mg. oxytetracycline upon antibiotic blood serum concentration.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
of both antibiotic (275 mg./l.) and TA salts also resulted in OTC serum level increases somewhat larger than those obtained with antibiotic and salts in the feed. A maximum response was suggested at 0.05% to 0.1% of TA salt. In this experiment, the ratio of water consumption to feed consumption was greater than 2:1; hence, intake of TA salts was greater by the groups receiving salts in the water than by those receiving salts in the feed. This difference may account for the relatively lower antibiotic serum levels obtained when both OTC and TA salts were feed administered. Comparison of the data shown in Figures 1 and 2 indicates that the degree of potentiation was greater with the higher level of antibiotic than with the lower, even though the range of TA salt levels was the same in the two series. No marked or consistent differences in weight gains or feed or water consumption due to administration of any level of
623
T E R E P H T H A L I C A C I D AND OXYTETRACYCLINE 3 HOURS POST INJECTION
zoo
6 HOURS POST INJECTION Control
Hi
16 0 Disodium i—i terephthalate '—'
"
80
-
r^ •
24 HOURS POST INJECTION
40
•
nfTTlTh
,•;., O
IO O IO O
N
O
O
O in O u> O O O M
es O no —
~"
CVJ
in N
O IO O — — CM
O m O m o o O N I) S O «l O
— —
Dose of disodium terephthalate-mg.
FIG. 4. The influence of disodium terephthalate given by subcutaneous injection concurrently with SO mg. oxytetracycline upon antibiotic blood serum concentration in chickens. still occurred to the higher levels of disodium terephthalate. Here, average serum concentrations were approximately 0.5 meg./ml. for the 150 and 200 mg. doses, as compared to 0.25 mcg./ml. for the control. No d a t a are exhibited for samples taken at 48 hours, since the majority did not show detectable antibiotic serum concentrations. Results obtained when O T C was injected at a higher dosage level are shown in Figure 4. Fifty 1 mg. antibiotic doses were given subcutaneously in 4.0 ml. total volume of solution, which also contained graded levels of disodium terephthalate. Antibiotic serum concentration in the control (non-potentiated) group at 3 hours was 12.3 mcg./ml. from 50 mg. antibiotic, as compared to 8.1 mcg./ml. obtained from the 25 mg. dose (Figure 3). I n contrast to the erratic 3-hour results obtained with the 25 mg. dose of antibiotic, all birds receiving 50 mg. antibiotic concurrently with disodium tereph-
thalate had markedly higher OTC serum concentrations a t 3 hours than did the control. Doses of the salt as low as 25 mg. per bird gave increases as great or greater t h a n 150 mg. and 200 mg. doses. By 6 hours post injection, the antibiotic serum concentration of the control had dropped to 8.7 mcg./ml., while concentrations in the groups injected with disodium terephthalate were maintained a t levels ranging from 12 to 16 mcg./ml. No relationship of potentiation response to T A salt dose was suggested. Moderate b u t consistent increases in serum antibiotic levels due to injection of disodium terephthalate were still apparent a t 24 hours post injection. Again, no effect of T A salt dosage level was apparent. At 48 hours post injection, m a n y samples did not show detectable O T C serum concentrations. D a t a for this time interval therefore are not given. The degree of potentiation at 6 hours somewhat exceeded t h a t obtained at 3
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
120
624
K. E. PRICE, Z. ZOLLI, J R . AND G. A. DONOVAN
and Zolli, 1959b). Although no explanation for this apparent anomaly is available at present, it may be that some property of the ester, such as its absorption or excretion rate, may account for the DISCUSSION observed variability in potentiating acPotentiation of oxytetracycline was tivity. In rats, preliminary findings have accomplished by oral and parenteral suggested that there are marked differadministration of various salts of tereph- ences between terephthalic acid and its thalic acid. Although data were too limited dimethyl ester with respect to the quanto be conclusive, there was an indication tity of the drug that can be found in the that solubility of a terephthalic compound urine following oral administration (Von may not be an important factor in its Fossen, 1959). Data on the excretion rate relative potentiating ability. Despite the of dimethyl terephthalate in chickens are extreme differences in the solubilities of not yet available. Limited experiments terephthalic acid and its derivatives have indicated that 15% to 25% of an (Table 3), activity of the acid was not orally administered dose of terephthalic shown to be appreciably less than that of acid can be recovered in the urine of the its much more soluble salts. Dimethyl- chicken (Price et al., unpublished data). terephthalate, which like TA, has only The results presented in this report slight solubility in water, had somewhat show that, by the use of soluble salts of less potentiating ability than the acid or terephthalic acid, oxytetracycline can be its salts. potentiated through the drinking water Despite the fact that the dimethyl as well as through the feed. In addition, ester of terephthalic acid was the least the data show that simultaneous paractive of all compounds tested, a sub- enteral administration of disodium terephstantial increase in OTC serum level was thalate and oxytetracycline provides achieved when this compound was fed higher antibiotic serum levels than concontinuously. This is of special interest, trols without a potentiating agent for at since it was previously found that simul- least twenty-four hours post injection. taneous injection of the ester with OTC Although data obtained were not preinto the ligated duodenal loop of chickens cise enough to define optimum levels of did not appreciably increase antibiotic the terephthalic acid derivatives, on the serum levels, whereas the acid did (Price average it appeared that near-maximum increases in antibiotic serum levels were TABLE 3.—Solubilities of terephthalic obtained with 0.05% to 0.15% concenacid and derivatives trations of the salts in the drinking water. Solubility in These concentrations are about | to f Compound water at 20°C, that required to produce near-maximum gm./lOO ml. 1 antibiotic serum level increases when adTerephthalic acid 0.0016 ministration of the salts was through the Dimethyl terephthalate <0.0016 Disodium terephthalate 7-10 feed. However, this would not necessarily Bis-monoethanolamine terephthalate 20-25 indicate greater efficacy of water-adminBis-triethanolamine terephthalate 20-25 Bis-diethanolamine terephthalate 30-35 istered salts, since the greater consump1 tion of water as compared to feed would As equivalent of free terephthalic acid.
hours with both levels of antibiotic when serum concentrations for each time interval resulting from all doses of TA salts were pooled.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
TEREPHTHALIC ACID AND OXYTETRACYCLINE
SUMMARY
This study concerned potentiation of serum concentrations of oxytetracycline (OTC) by terephthalic acid (TA), its sodium salt, and several of its soluble ethanolamine salts. Administration via water, feed and subcutaneous routes was investigated for both the antibiotic and the potentiating agents. The antibiotic was given at two levels, and the potentiating agents were administered at graded levels. Results show that TA and its soluble salts given by all three routes increase serum OTC levels of chickens over those of controls which received antibiotic only. When antibiotic was administered orally, degree of potentiation appeared to be greater with 500 gm./ton in feed, or 275 mg./l. in water, than it was with 200 gm./ton or 110 mg./l. This occurred whether TA salts were administered in feed or water. The data obtained did not permit determination of optimum levels of orally administered terephthalates for maximum potentiating activity. I t was indicated, however, that, with the levels -of OTC studied, maximum increases in serum
levels over controls would be obtained with 0.05% to 0.15% equivalent of free acid of soluble TA derivatives in water, and from two to four times this in feed. Because water intake was greater than feed intake, these percentages are equivalent to somewhat similar aggregate intakes by the two routes, and are not indicative of a greater effectiveness by one route as compared with the other. The data were not sufficient for estimating the optimum potentiating level of disodium terephthalate when administered subcutaneously. All doses definitely potentiated OTC serum levels, but those providing near maxima ranged from 25 mg. to 150 mg. per 1.5 pound bird under different conditions. The factor of time was studied with antibiotic and potentiating agents, both administered subcutaneously. I t was found that, on the average, the degree of potentiation at six hours post-injection was somewhat greater than at three hours with the doses used. ACKNOWLEDGMENT
The authors are grateful to Dr. A. P. Collins for the preparation of the terephthalate compounds used in this study and to Dr. D. F. Chichester for assistance in preparation of the manuscript. REFERENCES Grove, D. C , and W. A. Randall, 19SS. Assay Methods of Antibiotics, New York, Medical Encyclopedia, Inc. Price, K. E., Z. Zolli, Jr., J. C. Atkinson, A. P. Collins and H. G. Luther, 1959a. Antibiotic inhibitors. III. Reversal of calcium inhibition of intestinal absorption of oxytetracycline in chickens by certain acids and acid salts. Antibiotics Annual 1958-59, Medical Encyclopeida, Inc., New York, pp. 1020-1032. Price, K. E., Z. Zolli, Jr. and J. H. Hare, 1959b. Effect of dietary calcium-phosphorus adjustment and/or supplementation with terephthalic acid
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
probably result in a nearly comparable intake of TA salts by the two routes. Insufficient data are available for estimating the optimum level of disodium terephthalate required for maximum potentiation when this salt is administered parenterally. In some cases, doses as low as 25 mg. in a 1.5 pound bird were sufficient to yield near-maximum serum levels. In others, doses up to 150 mg. per bird were required for maximum potentiation. Despite this marked variation, there was a clearcut increase in OTC serum levels obtained with all dosages of disodium terephthalate.
625
626
K. E. PRICE, Z. ZOLLI, J R . AND G. A. DONOVAN
upon serum antibiotic levels of chickens. Poultry Sci. 38: 233-237. Price, K. E., and Z. Zolli, Jr., 1959a. The influence of dietary calcium-phosphorus and terephthalic acid on antibiotic control of experimental infectious synovitis. Avian Diseases, 3: 135-156.
Price, K. E., and Z. Zolli, Jr. 1959b. The influence of terephthalic acid on oxytetracycline serum levels in chickens. Studies on mode of action. I. Avian Diseases, 3: 157-169. Van Fossen, P., 1959. Personal communication.
PRAN VOHRA,* B. W. LANGER, JR. AND F. H. KRATZER Department of Poultry Husbandry, University of California, Davis (Received for publication August 31, 1959)
in excess than C14H? of methionine into methylaminoethanol. Young el al. (1955) found that choline requirement of chickens was decreased by dietary folic acid. Also, choline could be replaced by an equimolar quantity of monomethylaminoethanol plus betaine, either in presence or absence of folic acid. Vitamin BJ2 spared choline requirements of poults for optimum growth (Kratzer, 1952) and appeared to be involved in methyl synthesis (Vohra et al. 1956). In vitamin B^-depleted chicks and rats monomethylaminoethanol plus betaine were as effective as equimolar decarboxylation Serine > aminoethanol concentrations of dietary choline and methionine. However, vitamin Bi2 im"active HCHO" proved growth markedly if betaine con> N-hydroxymethylaminocentration was marginal in the ration reduction ethanol > methylaminoethanol (Young e/aZ., 1954,1957). In the present experiments, an attempt "active HCHO" > N-hydroxymethyl-N- has been made to investigate Stekol's hypothesis to throw some light on the reduction methylaminoethanol >• dimethyl- methyl acceptors for the biosynthesis of choline. "active methionine CH 3 " aminoethanol >
F
OR the biosynthesis of choline, Jukes (1947) and Artom (1952) proposed a scheme for the stepwise addition of methyl groups from methionine via transmethylation to aminoethanol. However, Stekol et al. (1955) demonstrated that dimethylaminoethanol was the only direct acceptor of the methyl group of methionine for choline biosynthesis in rats, the other 2 methyl groups were produced by de novo synthesis involving folic acid. Stekol (1958) postulated the following stepwise sequence for choline biosynthesis:
choline. The de novo synthesis of methyl groups was supported by studies of Nye (1956) in a mutant strain of Neurospora crassa where formate-C w was incorporated far * Present address: BOCM Poultry Demonstration Farm, Stoke Mandeville, Bucks, U.K.
EXPERIMENTAL
N-Hydroxymethylserine: The barium salt of N-hydroxymethylserine has been synthesized (Levene and Schormuller, 1934) but during drying of the product, it lost a molecule of water. The sodium salt of Nhydroxymethylserine was prepared essentially the same way. One mole of
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on April 17, 2015
Methyl Acceptors for the Biosynthesis of Choline