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Placental transfer of ampicillin
Placental transfer of ampicillin MALCOLM M.
A.
ABOU-SABE,
DAVID
CHARLES,
Pittsburgh,
Pennsylvania
MA~AULAY,
M.D.
PH.D.
M.B.,
M.R.C.O.G.
Subjects. The study was composed of 103 patients and the length of gestation varied from 15 to 40 weeks. Ampicillin was administered by intravenous injection in a dose of 250 mg. to 36 and a dose of 500 mg. to a further 53 patients. The compound was injected directly into the amniotic sac in a dose of 250 mg. on two occasions and 500 mg. to a further 10. Biological fluid samples. Control serum samples were obtained from pregnant and nonpregnant individuals who had not received any form of chemotherapy in the previous 3 months. Amniotic fluid for control studies was obtained prior to abdominal delivery or before the instillation of ampicillin into the amniotic sac. All samples of amniotic fluid submitted for assay for ampicillin were obtained by abdominal amniocentesis. Vaginal aspiration at the time of spontaneous rupture of the membrane in patients in active labor would result in contamination of amniotic fluid by maternal blood or vaginal microbial organisms which would invalidate the microbiologic assay procedures. Fetal blood was obtained from the umbilical vein at the time of delivery of the infant, but before completion of the third stage of labor. A uniform method of collection was devised by clamping a section of the umbilical cord and aspirating the blood from the umbilical vein. On no occasion was blood collected indiscriminantly from the umbilical cord by manual compression, as the latter produces a material composed of Wharton’s jelly and arterial and venous
DE s PITE THE recent advances in antibiotic therapy, intrauterine infection of the fetus is an ever present hazard of obstetric practice. Pryles and associates1 reported bacteriologically proved infection of the infant in 7 per cent of cases when the membranes had ruptured 12 to 15 hours prior to delivery. Our knowledge of placental transfer of compounds that could be beneficial to both mother and fetus is fragmentary. The present communication concerns the transfer of ampicillin across the human placenta and membranes. Material
and
methods
Compound. One of the major developments in the penicillin field has been the discovery that certain side chains render the penicillin molecule more active against gramnegative bacilli of the types encountered in the female genital tract. Ampicillin is a semisynthetic penicillin which has a relatively wide spectru.m of activity; this compound is 6- [ D (-) -alpha phenylacetamido] penicillanic acid, the presence of the phenyl glycine side chain distinguishes it from other penicillin analogs. In the present study the sodium salt, shown in Fig. 1, was used. The molecular weight is; 372.4. A parenteral solution of ampicillin was reconstituted from the powder by adding 2.5 ml. of sterile water to each 250 mg. ampule.
From the Uniuersitv of Pittsburgh Departments of Patholdgy, Obste%cs, and Gynecology, and Magee-Womens Hospital.
943
944
MacAulay,
Abou-Sabe,
and
Charles
SODIUM
SALT
AMPICILLIN
(-n) Ii
ii
Fig. 1. blood. Umbilical venous blood renders data more specific as it furnishes conclusive evidence of transfer from maternal to fetal circulation. All blood samples were allowed to clot and subsequent to centrifugation the serum was aseptically removed. All cellular elements were removed from the amniotic fluid samples by centrifugation. All fluid samples obtained were stored in a frozen state until the assays were performed. An accurate record was kept of the time the ampicillin was administered and the biologic fluid samples obtained. Amniotic fluid volumes were determined by the P.A.H. technique of Charles and Jacobv.2 Assay procedures. A modification of the cylinder-plate method was used. The test organism Sarcina lutea, ATCC 10054, was maintained on nutrient agar. To obtain Sarcina for the seed layer, transfers were made to nutrient agar slants incubated for 8 hours. These slants were transferred to a second series of slants incubated for 18 hours, and all actively growing organisms were washed off with distilled water. This suspension was diluted to give 50 per cent light transmission in a Bausch and Lomb spectrophotometer set to pass a wave length of 650 mp. From the standardized suspension the seed layer was inoculated in a proportion of 0.25 ml. of suspension to 100 ml. of seed cooled to 5o” c.
In the preparation of the plates 10 ml. of base agar was evenly distributed in 2.0 y 10.0 cm. Petri dishes and allowed to harden. Four milliliters of the inoculated seed layer was spread over the base agar and allowed to set. Six stainless steel cylinders (internal diameter 0.6 cm., length 1.0 cm. ) were placed on the seed layer at 60 degree intervals on a circle of 2.8 cm. radius. Standard solutions of the antibiotic and biologic &lids for assay were added to the cylinders in 0.25 ml. quantities. The plates were covered with porcelain tops and incubated for 18 to 24 hours at 34 to 37O C. Each specimen assayed was added to three cylinders on each of three plates. The other three cylinders of every plate contained a known concentration of ampicillin in the same type of fluid as that to be assayed. Thereby, three cylinders were available to correct for individual plate deviations from the inhibition expected from a standard curve obtained from a series of twelve plates. The standard curve was obtained using known concentrations of 0.66, 1.25, 2.50, and 5.0 yg per milliliter of ampicillin. At least 9 cylinders were used for each concentration. The reference concentration level was 1.25 ,bg per milliliter and up to 200 cylinders were available for this average determination. The diameter of the average zone of inhibition was plotted on the logarithmic scale against concentrations on the
Volume Number
96 7
Placental
linear side of semilogarithmic paper. The position of the zones of inhibition on this curve allowed the concentration in the fluid assayed to be determined. Experiments and results To obtain a serum concentration versus time relationship in normal nonpregnant subjects for comparison to future experiments, and to verify the effectiveness of the assay procedure, 250 mg. of ampicillin was administered intravenously to a nonpregnant female and to a normal male. Blood samples were collected over a 4 hour period and assayed. The results are plotted in Fig. 2; the loss of antibiotic in both subjects shows
transfer
of
ampicillin
945
the same time relationship. It will be noted that within 3yz hours the serum levels have fallen to less than 1 pg from an initial level of 17 pg per milliliter. Eighteen patients in active labor received 250 mg. ampicillin by intravenous injection and the assay results of the fetal and maternal sera are shown in Fig. 3. No differentiation between maternal and fetal concentration-time relationships can be reliably determined; in some instances maternal levels were higher than fetal and vice versa. Only at the earlier times were maternal levels consistently higher than the corresponding fetal levels. In both fetus and mother, the level of ampicillin remained above 1 rg per milli-
24r-rl
24
22
I
I
I
I
I
1
1
1
1
-
20 0
NON
A
NORMAL
FEMALE
PREGNANT
-
MALE
18
0
MATERNAL
A
CORD
SERUM
SERUM
I6
I4
12
IO I P
I
2 HOURS
Fig.
2.
3
4 AFTER
5
6
7
250MM
6
s
IO
3
II
I.V.
HOURS AFTER INJECTION OF 250 WY I.V. TO MOTHER Fig.
3.
946
MacAulay,
Abou-Sabe,
24
I
I
I
and
I
I
Charles
I
I
I
I
22
20
M n
18
MATERMALSERWY COROSERUM
16
1 -4
14
12
IO
) 8
! 6
I
2
3
4
5
6
7
8
9
HOURS AFTER INJECTION OF 5OOMeM I.V. TO MOTHER Fig.
4.
Table I. Amniotic into __-
amniotic
fluid
levels of ampicillin
IO
II
‘IThirteen patients -\vere gi\:en X0 ~rq. oi ampicillin intra\:enously and the assa\’ I’Psuits are depicted in Fig. 4. With this increased dose the maternal levels werr consistently higher than fetal, the difference initially being more marked than that of the previous experiment and the sustained Ievels beyond 3 hours were again evident. 4 further 10 patients received 500 my. of ampicillin by intravenous injection and serum and amniotic fluid levels were determined at intervals up to 5 hours. Control amniotit fluid and subsequent samples were obtained by amniocentesis. The results arc shown in Fig. 5. The maternal serum values fell on the curve of expected values which is projected as dashes in Fig. 5. Detectable levels were not obtained in amniotic fluid until approximately 2 hours after drug administration. The limit of sensitivity of the bioassay with the organism used, under the conditions of each assay, varied from 0.4 to 0.7 pug per milliliter. To determine Lvhether lower levels might be present in the samples obtained within the first 2 hours, aliquots and control amniotic fluid were lyophilized, rehydrated to give sufficient material and reassayed. After lyophilization control fluid caused some inhibition of growth of the assay organism. Allowing for this inhibition the concentrating procedure permitted the detection of original fluid levels as low as
35 minutes
after
injection
of 500 mg.
sac Concentration
Weeks’ gesftftion
Patient c. 0.
L.
15
Temainiflg in
amniotic fluid (m&ml.)
Amniotic fluid volume (ml.)
0.162
280
Total amount 7emaining
(mk5) 33.6
Maternal Per
cent
remaining 6.7
serum
(w/ml.) 39.0
Remarks Rubella,
first
tti-
mester
D. A. R. H. E. S. W. A. D.
28 31 33
0.375 0.580 0.325
878 900 600
312.0 522.0 195.0
63.0 100.0 39.0
Normal
preg-
nancy sensitization
Rh Normal nancy ---
preg.---
Volume
96
Number
7
Placental
I
2
3
4
HOURS AFTER 500 MILLIGRAMS
5
6
transfer
of
ampicillin
7
8
7
0
947
INJECTION OF AMPICILLIN I.V.
Fig. 5.
I HOURS
2
3
4
5
6
AFTER INJECTION OF 500 YILLIORAMS AMPICILLIN INTO AMNIOTIC FLUID
Fig. 6. 0.07 pg per milliliter; within the first hour no samples had levels exceeding this concentration. One patient at term had 500 mg. of ampicillin injected into the amniotic sac and maternal serum levels were followed for 5 hours. The results of this experiment are shown in Fig. 6. After the initial slow rise
it is evident that the serum concentration remained at approximately 2 rg per milliliter for the entire period. The fetal serum level at the time of delivery is also shown in Fig. 6; it was fully five times as high as the maternal level at delivery. Four patients had amniotic fluid volumes determined and 500 mg. of ampicillin in-
948
MacAulay,
Abou-Sabe,
and Charles
jetted into the amniotic sac. The amniotic fluid ampicillin levels determined 35 minutes following injection are shown in Table 1. The 2 patients with normal pregnancies in the third trimester had 63 per cent and 39 per cent of the ampicillin remaining in the amniotic fluid after 35 minutes; they contrast markedly with the patient in the early part of the second trimester who had only 6.7 per cent remaining after 35 minutes. It will be noted that the maternal serum levels can be correlated with the lower and higher transfer rates found in late and early pregnancy. Comment
In the evaluation of the biologic assay it is necessary to consider procedures, whether substances other than penicillin could account for inhibition of the growth of Sarcirza lutea and so produce false positive or elevated levels of antibiotic. Very early in the study several untreated samples of serum and amniotic fluid were found to inhibit the growth of E. coli, Staphytococcus aureus, Sarcina lutea, and several other organisms when added to an inoculum in broth or to cylinders in the cylinder-plate method. The inhibitory effect was, however, inactivated by heating at 56’ C. for 30 minutes in a water bath. Such treatment did not destroy ampicillin in distilled water or samples of physiologic fluid. Penicillinase was found to negate inhibition by inactivated serums from patients given ampicillin. Pursuant to these findings, all samples were inactivated prior to assay. The control amniotic fluids obtained prior to drug administration were assayed with the treated samples. Untreated maternal serums were also added to the plates, but as it was impossible to obtain control fetal serum, we had to rely on inactivation having been effected by heat. Para-aminohippurate, in concentrations used for volume measurements, did not inhibit the growth of Sarcina lutea. One of the possibilities for the identity of the inhibitory activity of some sera and amniotic fluids is a complement-dependent antibody, as it is very well known that both
the end-piccc ( C’l ) and mid-piece <-‘E: 01 the complemnlt are inactivated hv hwting for a frw minutes at 06” C:. ‘I’hc inhibitory activity of at 1o.i~~ anti amniotic fluid was concentration-dependent. as lyophilization of an aliquot of 10 C.C. and reconstitution of the original volume by 1 ml. increments resulted in a linear rclationship between the concentration effected ancl the zone of inhibition of growth. The incidence of this activity in amniotic fluids is not known, but we ha\re found it in 2 cases from the 16 studied in this series. Walsh, Hildebrandt, and Prystowsky’ found no inhibition in 5 amniotic fluid samples studied for support of growth of a number of clinically important microorganisms. Normal serum or amniotic fluid without antibacterial activity is the logical diluent for bioassay procedures of this type as they reflect distribution of the drug in viva, and as a result these were used as our standard of reference. By the simultaneous use of suitable untreated controls, with fluids being assayed, and inactivation for 30 minutes at 57’ C.. it has been possible to rule out artifacts in the determination of ampicillin levels by the cylinder-plate method. From the results shown in Figs. 3 and 4, it is obvious that one of the factors affecting both maternal and fetal levels of antibiotic is dose. In preliminary studies using oral administration of 500 mg. every 6 hours, the fetal concentration remained one quarter of that in the maternal serum. The results were inconsistent possibly due to factors invoiving ,y:astrointestinal absorption in late pregnancy.“, Ci In subsequent studies the antibiotic was administered parenterally in order to rule out possible variables that would affect the apparent transfer to the fetus. In comparing the 250 and 500 mg. intravenous doses, it is apparent that after 1 hour the fetal concentration is about 7 jhg per milliliter in both cases. At periods later than 6 hours after 250 mg. intravenously, the levels in both mother and fetus are below 1 pg per milliliter, as indicated by the dotted line in Fig. 3. After 500 mg. intravenously, however, the levels at 6 hours are approxi-
Volume Number
96 7
mately 2 pg per milliliter. If ampicillin is to be used to prevent intrauterine infections, levels of 5 pg per milliliter or more will probably have to be maintained in order to affect minimum inhibitory concentration for E. coli, but levels of greater than 2 ,ug per milliliter, will probably be sufficient as minimum inhibitory concentrations for other potential pathogenic organisms inhabiting the vagina. 7, * Work is in progress to determine if a 4, hour intravenous regimen or oral administration with probenecid will sustain sufficient serum levels. Eickhoff, Kislak, and Finland” have reported higher, more sustained serum levels of ampicillin with administration of probenecid. In view of the low minimal inhibitory concentration for the gram-negative organisms relative to the other penicillins, it is likely that a suitable dosage schedule can be obtained without resorting to the high dosage used by Weinstein, Lerner, and Chewlo in the treatment of infections caused by gram-negative bacilli. The large doses used by the latter authorslO were not toxic to adults, but there is a theoretical possibility that very high serum levels of some penicillin analogs can have a toxic effect on the fetus and neonate by releasing bilirubin bound to the serum proteins. Odellll has found that certain sulfonamides release bilirubin from serum proteins. Kunin12 has found that there is competition for protein-binding sites between some penicillins and sulfonamides. Hence it is possible that the bilirubin sulfonamides, and penicillin-binding sites are the same and that high levels of penicillin may effect a release of bilirubin from serum proteins and so increase any risk of kernicterus. Ampicillin is one of the least protein-bound penicillinsl’ and, .thus, is less likely to have this theoretical d.anger. Penicillin G, dimethoxypenicillin, and oxacillin all pass from mother to fetusI l** l5; these antibiotics do not have as wide a spectrum of action as ampicillin and are more highly protein bound, with the result that they are less desirable for use in the prevention of intrauterine infection. Several antibiotics are suspect as thera-
Placental
transfer
of
ampicillin
949
peutic agents in pregnancy. Tetracyclines have a destructive effect on development of deciduous teethlO and bone gr0wth.l’ Streptomycin has a deleterious effect on the fetal ear.l* The administration of chloramphenicol has been associated with the “gray infant syndrome.“lS Cephalosporin has a wide spectrum of activity and passes to the fetus.?” Since there is an apparent absence of crossreaction in patients sensitized to penicillin, it may prove effective in the prevention of intrauterine infection in patients sensitive to ampicillin. The finding that the maternal and fetal levels remained slightly higher than nonpregnant controls after 4 hours (Figs. 2, 3, and 4) suggested that the amniotic fluid compartment might be acting as a reservoir or depot into which the antibiotic was being sequestered and then slowly released. The experimental results shown in Figs. 5 and 6 support this suggestion. The antibiotic does not appear in the amniotic fluid in detectable quantities until after 1 hour, and the level increases continuously as the materna1 concentration falls. Six hours after intravenous injection the amniotic fluid level is approximately twice the maternal or fetal serum level. When the antibiotic is injected into the amniotic sac there is a slow release into the maternal serum, with maternal levels becoming constant after 90 minutes. The fetal level is very high at delivery after 6 hours. It is possible that the transfer to the mother is effected in large measure through the fetus, although the findings in no way differentiate between this and transfer through the amnion over the chorion frondosum with relatively more of the antibiotic being carried back to the fetus through chorionic vessels and less transferring through the intervillous sinus to the maternal blood. It is very interesting to note that the passage out of the amniotic fluid is much more rapid in early pregnancy. As shown in Table I, Patient C. 0. L. at 15 weeks’ gestation transferred 93 per cent of a 500 mg. injection out of the sac in 35 minutes. There are too many variables involved here to
950
MacAulay,
Abou-Sabe,
and
Charles
warrant any suggestion as to the origin of the finding, further study is needed to decide Lvhether the greater efficiency is a function of early pregnancy or whether the effect is due to pathophysiology resulting from rubella. The same comment may be made of Patient H. E. S., whose placenta and membranes had transferred virtually no antibiotic in 35 minutes. The 4 cases of Table I raise the possibilities that ( 1’) in earlier pregnancy the system of fetus, amnion, chorion laeve, chorion frondosum. and decidua transfer ampicillin much more rapidly; (2) the changes in the system near term decrease the efficiency for passage of ampicillin: (3 I rubella affects the system in such a \vay as to cause increased transfer of ampicillin; (4) Rh sensitization diminishes the capacity of the system for the transference of ampicillin. In studies” where ampicillin was administered by mouth, inconsistent levels were cncountertxd in the amniotic fluid. The authors included, without comment. 4 patients with Rh sensitization: low levels of antibiotic were encountered in fluid ob-
tained by amniocenteses. ‘These result<. obtained by transfer in a direction opposite to the one studied in Patient H. E. S.. arcs consistent with the proposition that transfer rat.es art: diminished in the Rh-sensitizrd state. Summary
Parameters of the transference of ampicillin among the three compartments I mother. fetus: and amniotic sac) have been studied in 103 patients. A rapid transfer occurs from the mother to the fetus, with fetal serum levels reaching levels exceeding rninimal inhibitory concentrations for many organisms causing intrauterine infections. The antibiotic appears in the amniotic fluid after 90 minutes; the amniotic fluid compartment appears to form a depot for, tht, sequestration of ampicillin. Varying ratrs of transfer out of the amniotic fluid arf’ found in different stages of pregnancy and pathologic conditions. The findina of a heat-labile system, inhibitory for the ,qrowth of Sarcirzo li~tczu, is recorded.
REFERENCES
Prylcs, C. V., Steg, N. L., Nair, S., Gellis, S. S.. and Tenney, B.: Pediatrics 31: 608, 1963. Charles, D., and Jacoby, H. E.: .4~. J. OBST. & GYNEC. In press, 1966. Ferrata, A.: Berlin klin. Wschr. 44: 366, 1907. Walsh, H., Hildebrandt, R. J., and Prystowsky, H.: Ahf. J. OBST. & GYNEC. 93: 590, 5. 6. 7. 8. 9. to. Il.
12. 13. 14. 15.
Kunin, C. M.: Proc. Sot. Exper. Biol. B Med. 117: 69, 1964. Charles, D.: J. Obst. & Gynaec. Brit. Emp. 61: 750, 1954. Bunn. P., Knight, R., and Amberg. J.: New York J. Med. 60: 3074, 1960. Prigot, A., Froix, C. J.. and Rubin, E.: Anti-
1965.
microb.
Elliott. R. B., Stokes, E. ,J., and Maxwell, G. M.: Arch. Dis. Childhood 39: 101, 1964. Belcher. T. E., Edgar, W. M., Melville, H. A. H., and Peel, K. R.: Brit. M. J. 1: 137: 1966. Rolinson, G. N.. and Stevens, S.: Brit. M. J. 2: 191, 1961. Quinn. E. L., and Cox, F.: Antimicrob. Agents & Chemotherap., 1963, p. 639. Eickhoff, T. C., Kislak, J. W., and Finland, M.: ,4m. J. M. SC. 249: 183, 1965. Weinstein. L.. Lerner. P. I.. and Chew. W. H.: New ‘E&land J. ‘Med. ‘271: 525, i964. Odell. G. B.: J. Pediat. 55: 268, 1959.
402. Kline, A. H., Blattner, R. J., and Lunin, 1. A. M. il. 188: 170. 1964. kohlan, S. Q., Beveiander, G., and Broz. i\ntirnicrob. Agents Pr Chemotherap.. p. 340. Conway, N., and Birt, B. D.: Brit. M. 260. 1965. Lisdhner, H., Seligman, S. T.. Krammer, and Parmelee. A. H.: 1. Pediat. 59: 21. Barber, M., Bnd Waterworth, P. M.: M. J. 2: 344, 1964.
16. 17.
18. 19. 20.
Agents
& Chcmotherap..
1962, p. M.: S : 1961. J. 2: A.. 1961. Brit.
A.
ABOU-SABE,
DAVID
CHARLES,
Pittsburgh,
Pennsylvania
MA~AULAY,
M.D.
PH.D.
M.B.,
M.R.C.O.G.
Subjects. The study was composed of 103 patients and the length of gestation varied from 15 to 40 weeks. Ampicillin was administered by intravenous injection in a dose of 250 mg. to 36 and a dose of 500 mg. to a further 53 patients. The compound was injected directly into the amniotic sac in a dose of 250 mg. on two occasions and 500 mg. to a further 10. Biological fluid samples. Control serum samples were obtained from pregnant and nonpregnant individuals who had not received any form of chemotherapy in the previous 3 months. Amniotic fluid for control studies was obtained prior to abdominal delivery or before the instillation of ampicillin into the amniotic sac. All samples of amniotic fluid submitted for assay for ampicillin were obtained by abdominal amniocentesis. Vaginal aspiration at the time of spontaneous rupture of the membrane in patients in active labor would result in contamination of amniotic fluid by maternal blood or vaginal microbial organisms which would invalidate the microbiologic assay procedures. Fetal blood was obtained from the umbilical vein at the time of delivery of the infant, but before completion of the third stage of labor. A uniform method of collection was devised by clamping a section of the umbilical cord and aspirating the blood from the umbilical vein. On no occasion was blood collected indiscriminantly from the umbilical cord by manual compression, as the latter produces a material composed of Wharton’s jelly and arterial and venous
DE s PITE THE recent advances in antibiotic therapy, intrauterine infection of the fetus is an ever present hazard of obstetric practice. Pryles and associates1 reported bacteriologically proved infection of the infant in 7 per cent of cases when the membranes had ruptured 12 to 15 hours prior to delivery. Our knowledge of placental transfer of compounds that could be beneficial to both mother and fetus is fragmentary. The present communication concerns the transfer of ampicillin across the human placenta and membranes. Material
and
methods
Compound. One of the major developments in the penicillin field has been the discovery that certain side chains render the penicillin molecule more active against gramnegative bacilli of the types encountered in the female genital tract. Ampicillin is a semisynthetic penicillin which has a relatively wide spectru.m of activity; this compound is 6- [ D (-) -alpha phenylacetamido] penicillanic acid, the presence of the phenyl glycine side chain distinguishes it from other penicillin analogs. In the present study the sodium salt, shown in Fig. 1, was used. The molecular weight is; 372.4. A parenteral solution of ampicillin was reconstituted from the powder by adding 2.5 ml. of sterile water to each 250 mg. ampule.
From the Uniuersitv of Pittsburgh Departments of Patholdgy, Obste%cs, and Gynecology, and Magee-Womens Hospital.
943
944
MacAulay,
Abou-Sabe,
and
Charles
SODIUM
SALT
AMPICILLIN
(-n) Ii
ii
Fig. 1. blood. Umbilical venous blood renders data more specific as it furnishes conclusive evidence of transfer from maternal to fetal circulation. All blood samples were allowed to clot and subsequent to centrifugation the serum was aseptically removed. All cellular elements were removed from the amniotic fluid samples by centrifugation. All fluid samples obtained were stored in a frozen state until the assays were performed. An accurate record was kept of the time the ampicillin was administered and the biologic fluid samples obtained. Amniotic fluid volumes were determined by the P.A.H. technique of Charles and Jacobv.2 Assay procedures. A modification of the cylinder-plate method was used. The test organism Sarcina lutea, ATCC 10054, was maintained on nutrient agar. To obtain Sarcina for the seed layer, transfers were made to nutrient agar slants incubated for 8 hours. These slants were transferred to a second series of slants incubated for 18 hours, and all actively growing organisms were washed off with distilled water. This suspension was diluted to give 50 per cent light transmission in a Bausch and Lomb spectrophotometer set to pass a wave length of 650 mp. From the standardized suspension the seed layer was inoculated in a proportion of 0.25 ml. of suspension to 100 ml. of seed cooled to 5o” c.
In the preparation of the plates 10 ml. of base agar was evenly distributed in 2.0 y 10.0 cm. Petri dishes and allowed to harden. Four milliliters of the inoculated seed layer was spread over the base agar and allowed to set. Six stainless steel cylinders (internal diameter 0.6 cm., length 1.0 cm. ) were placed on the seed layer at 60 degree intervals on a circle of 2.8 cm. radius. Standard solutions of the antibiotic and biologic &lids for assay were added to the cylinders in 0.25 ml. quantities. The plates were covered with porcelain tops and incubated for 18 to 24 hours at 34 to 37O C. Each specimen assayed was added to three cylinders on each of three plates. The other three cylinders of every plate contained a known concentration of ampicillin in the same type of fluid as that to be assayed. Thereby, three cylinders were available to correct for individual plate deviations from the inhibition expected from a standard curve obtained from a series of twelve plates. The standard curve was obtained using known concentrations of 0.66, 1.25, 2.50, and 5.0 yg per milliliter of ampicillin. At least 9 cylinders were used for each concentration. The reference concentration level was 1.25 ,bg per milliliter and up to 200 cylinders were available for this average determination. The diameter of the average zone of inhibition was plotted on the logarithmic scale against concentrations on the
Volume Number
96 7
Placental
linear side of semilogarithmic paper. The position of the zones of inhibition on this curve allowed the concentration in the fluid assayed to be determined. Experiments and results To obtain a serum concentration versus time relationship in normal nonpregnant subjects for comparison to future experiments, and to verify the effectiveness of the assay procedure, 250 mg. of ampicillin was administered intravenously to a nonpregnant female and to a normal male. Blood samples were collected over a 4 hour period and assayed. The results are plotted in Fig. 2; the loss of antibiotic in both subjects shows
transfer
of
ampicillin
945
the same time relationship. It will be noted that within 3yz hours the serum levels have fallen to less than 1 pg from an initial level of 17 pg per milliliter. Eighteen patients in active labor received 250 mg. ampicillin by intravenous injection and the assay results of the fetal and maternal sera are shown in Fig. 3. No differentiation between maternal and fetal concentration-time relationships can be reliably determined; in some instances maternal levels were higher than fetal and vice versa. Only at the earlier times were maternal levels consistently higher than the corresponding fetal levels. In both fetus and mother, the level of ampicillin remained above 1 rg per milli-
24r-rl
24
22
I
I
I
I
I
1
1
1
1
-
20 0
NON
A
NORMAL
FEMALE
PREGNANT
-
MALE
18
0
MATERNAL
A
CORD
SERUM
SERUM
I6
I4
12
IO I P
I
2 HOURS
Fig.
2.
3
4 AFTER
5
6
7
250MM
6
s
IO
3
II
I.V.
HOURS AFTER INJECTION OF 250 WY I.V. TO MOTHER Fig.
3.
946
MacAulay,
Abou-Sabe,
24
I
I
I
and
I
I
Charles
I
I
I
I
22
20
M n
18
MATERMALSERWY COROSERUM
16
1 -4
14
12
IO
) 8
! 6
I
2
3
4
5
6
7
8
9
HOURS AFTER INJECTION OF 5OOMeM I.V. TO MOTHER Fig.
4.
Table I. Amniotic into __-
amniotic
fluid
levels of ampicillin
IO
II
‘IThirteen patients -\vere gi\:en X0 ~rq. oi ampicillin intra\:enously and the assa\’ I’Psuits are depicted in Fig. 4. With this increased dose the maternal levels werr consistently higher than fetal, the difference initially being more marked than that of the previous experiment and the sustained Ievels beyond 3 hours were again evident. 4 further 10 patients received 500 my. of ampicillin by intravenous injection and serum and amniotic fluid levels were determined at intervals up to 5 hours. Control amniotit fluid and subsequent samples were obtained by amniocentesis. The results arc shown in Fig. 5. The maternal serum values fell on the curve of expected values which is projected as dashes in Fig. 5. Detectable levels were not obtained in amniotic fluid until approximately 2 hours after drug administration. The limit of sensitivity of the bioassay with the organism used, under the conditions of each assay, varied from 0.4 to 0.7 pug per milliliter. To determine Lvhether lower levels might be present in the samples obtained within the first 2 hours, aliquots and control amniotic fluid were lyophilized, rehydrated to give sufficient material and reassayed. After lyophilization control fluid caused some inhibition of growth of the assay organism. Allowing for this inhibition the concentrating procedure permitted the detection of original fluid levels as low as
35 minutes
after
injection
of 500 mg.
sac Concentration
Weeks’ gesftftion
Patient c. 0.
L.
15
Temainiflg in
amniotic fluid (m&ml.)
Amniotic fluid volume (ml.)
0.162
280
Total amount 7emaining
(mk5) 33.6
Maternal Per
cent
remaining 6.7
serum
(w/ml.) 39.0
Remarks Rubella,
first
tti-
mester
D. A. R. H. E. S. W. A. D.
28 31 33
0.375 0.580 0.325
878 900 600
312.0 522.0 195.0
63.0 100.0 39.0
Normal
preg-
nancy sensitization
Rh Normal nancy ---
preg.---
Volume
96
Number
7
Placental
I
2
3
4
HOURS AFTER 500 MILLIGRAMS
5
6
transfer
of
ampicillin
7
8
7
0
947
INJECTION OF AMPICILLIN I.V.
Fig. 5.
I HOURS
2
3
4
5
6
AFTER INJECTION OF 500 YILLIORAMS AMPICILLIN INTO AMNIOTIC FLUID
Fig. 6. 0.07 pg per milliliter; within the first hour no samples had levels exceeding this concentration. One patient at term had 500 mg. of ampicillin injected into the amniotic sac and maternal serum levels were followed for 5 hours. The results of this experiment are shown in Fig. 6. After the initial slow rise
it is evident that the serum concentration remained at approximately 2 rg per milliliter for the entire period. The fetal serum level at the time of delivery is also shown in Fig. 6; it was fully five times as high as the maternal level at delivery. Four patients had amniotic fluid volumes determined and 500 mg. of ampicillin in-
948
MacAulay,
Abou-Sabe,
and Charles
jetted into the amniotic sac. The amniotic fluid ampicillin levels determined 35 minutes following injection are shown in Table 1. The 2 patients with normal pregnancies in the third trimester had 63 per cent and 39 per cent of the ampicillin remaining in the amniotic fluid after 35 minutes; they contrast markedly with the patient in the early part of the second trimester who had only 6.7 per cent remaining after 35 minutes. It will be noted that the maternal serum levels can be correlated with the lower and higher transfer rates found in late and early pregnancy. Comment
In the evaluation of the biologic assay it is necessary to consider procedures, whether substances other than penicillin could account for inhibition of the growth of Sarcirza lutea and so produce false positive or elevated levels of antibiotic. Very early in the study several untreated samples of serum and amniotic fluid were found to inhibit the growth of E. coli, Staphytococcus aureus, Sarcina lutea, and several other organisms when added to an inoculum in broth or to cylinders in the cylinder-plate method. The inhibitory effect was, however, inactivated by heating at 56’ C. for 30 minutes in a water bath. Such treatment did not destroy ampicillin in distilled water or samples of physiologic fluid. Penicillinase was found to negate inhibition by inactivated serums from patients given ampicillin. Pursuant to these findings, all samples were inactivated prior to assay. The control amniotic fluids obtained prior to drug administration were assayed with the treated samples. Untreated maternal serums were also added to the plates, but as it was impossible to obtain control fetal serum, we had to rely on inactivation having been effected by heat. Para-aminohippurate, in concentrations used for volume measurements, did not inhibit the growth of Sarcina lutea. One of the possibilities for the identity of the inhibitory activity of some sera and amniotic fluids is a complement-dependent antibody, as it is very well known that both
the end-piccc ( C’l ) and mid-piece <-‘E: 01 the complemnlt are inactivated hv hwting for a frw minutes at 06” C:. ‘I’hc inhibitory activity of at 1o.i~~ anti amniotic fluid was concentration-dependent. as lyophilization of an aliquot of 10 C.C. and reconstitution of the original volume by 1 ml. increments resulted in a linear rclationship between the concentration effected ancl the zone of inhibition of growth. The incidence of this activity in amniotic fluids is not known, but we ha\re found it in 2 cases from the 16 studied in this series. Walsh, Hildebrandt, and Prystowsky’ found no inhibition in 5 amniotic fluid samples studied for support of growth of a number of clinically important microorganisms. Normal serum or amniotic fluid without antibacterial activity is the logical diluent for bioassay procedures of this type as they reflect distribution of the drug in viva, and as a result these were used as our standard of reference. By the simultaneous use of suitable untreated controls, with fluids being assayed, and inactivation for 30 minutes at 57’ C.. it has been possible to rule out artifacts in the determination of ampicillin levels by the cylinder-plate method. From the results shown in Figs. 3 and 4, it is obvious that one of the factors affecting both maternal and fetal levels of antibiotic is dose. In preliminary studies using oral administration of 500 mg. every 6 hours, the fetal concentration remained one quarter of that in the maternal serum. The results were inconsistent possibly due to factors invoiving ,y:astrointestinal absorption in late pregnancy.“, Ci In subsequent studies the antibiotic was administered parenterally in order to rule out possible variables that would affect the apparent transfer to the fetus. In comparing the 250 and 500 mg. intravenous doses, it is apparent that after 1 hour the fetal concentration is about 7 jhg per milliliter in both cases. At periods later than 6 hours after 250 mg. intravenously, the levels in both mother and fetus are below 1 pg per milliliter, as indicated by the dotted line in Fig. 3. After 500 mg. intravenously, however, the levels at 6 hours are approxi-
Volume Number
96 7
mately 2 pg per milliliter. If ampicillin is to be used to prevent intrauterine infections, levels of 5 pg per milliliter or more will probably have to be maintained in order to affect minimum inhibitory concentration for E. coli, but levels of greater than 2 ,ug per milliliter, will probably be sufficient as minimum inhibitory concentrations for other potential pathogenic organisms inhabiting the vagina. 7, * Work is in progress to determine if a 4, hour intravenous regimen or oral administration with probenecid will sustain sufficient serum levels. Eickhoff, Kislak, and Finland” have reported higher, more sustained serum levels of ampicillin with administration of probenecid. In view of the low minimal inhibitory concentration for the gram-negative organisms relative to the other penicillins, it is likely that a suitable dosage schedule can be obtained without resorting to the high dosage used by Weinstein, Lerner, and Chewlo in the treatment of infections caused by gram-negative bacilli. The large doses used by the latter authorslO were not toxic to adults, but there is a theoretical possibility that very high serum levels of some penicillin analogs can have a toxic effect on the fetus and neonate by releasing bilirubin bound to the serum proteins. Odellll has found that certain sulfonamides release bilirubin from serum proteins. Kunin12 has found that there is competition for protein-binding sites between some penicillins and sulfonamides. Hence it is possible that the bilirubin sulfonamides, and penicillin-binding sites are the same and that high levels of penicillin may effect a release of bilirubin from serum proteins and so increase any risk of kernicterus. Ampicillin is one of the least protein-bound penicillinsl’ and, .thus, is less likely to have this theoretical d.anger. Penicillin G, dimethoxypenicillin, and oxacillin all pass from mother to fetusI l** l5; these antibiotics do not have as wide a spectrum of action as ampicillin and are more highly protein bound, with the result that they are less desirable for use in the prevention of intrauterine infection. Several antibiotics are suspect as thera-
Placental
transfer
of
ampicillin
949
peutic agents in pregnancy. Tetracyclines have a destructive effect on development of deciduous teethlO and bone gr0wth.l’ Streptomycin has a deleterious effect on the fetal ear.l* The administration of chloramphenicol has been associated with the “gray infant syndrome.“lS Cephalosporin has a wide spectrum of activity and passes to the fetus.?” Since there is an apparent absence of crossreaction in patients sensitized to penicillin, it may prove effective in the prevention of intrauterine infection in patients sensitive to ampicillin. The finding that the maternal and fetal levels remained slightly higher than nonpregnant controls after 4 hours (Figs. 2, 3, and 4) suggested that the amniotic fluid compartment might be acting as a reservoir or depot into which the antibiotic was being sequestered and then slowly released. The experimental results shown in Figs. 5 and 6 support this suggestion. The antibiotic does not appear in the amniotic fluid in detectable quantities until after 1 hour, and the level increases continuously as the materna1 concentration falls. Six hours after intravenous injection the amniotic fluid level is approximately twice the maternal or fetal serum level. When the antibiotic is injected into the amniotic sac there is a slow release into the maternal serum, with maternal levels becoming constant after 90 minutes. The fetal level is very high at delivery after 6 hours. It is possible that the transfer to the mother is effected in large measure through the fetus, although the findings in no way differentiate between this and transfer through the amnion over the chorion frondosum with relatively more of the antibiotic being carried back to the fetus through chorionic vessels and less transferring through the intervillous sinus to the maternal blood. It is very interesting to note that the passage out of the amniotic fluid is much more rapid in early pregnancy. As shown in Table I, Patient C. 0. L. at 15 weeks’ gestation transferred 93 per cent of a 500 mg. injection out of the sac in 35 minutes. There are too many variables involved here to
950
MacAulay,
Abou-Sabe,
and
Charles
warrant any suggestion as to the origin of the finding, further study is needed to decide Lvhether the greater efficiency is a function of early pregnancy or whether the effect is due to pathophysiology resulting from rubella. The same comment may be made of Patient H. E. S., whose placenta and membranes had transferred virtually no antibiotic in 35 minutes. The 4 cases of Table I raise the possibilities that ( 1’) in earlier pregnancy the system of fetus, amnion, chorion laeve, chorion frondosum. and decidua transfer ampicillin much more rapidly; (2) the changes in the system near term decrease the efficiency for passage of ampicillin: (3 I rubella affects the system in such a \vay as to cause increased transfer of ampicillin; (4) Rh sensitization diminishes the capacity of the system for the transference of ampicillin. In studies” where ampicillin was administered by mouth, inconsistent levels were cncountertxd in the amniotic fluid. The authors included, without comment. 4 patients with Rh sensitization: low levels of antibiotic were encountered in fluid ob-
tained by amniocenteses. ‘These result<. obtained by transfer in a direction opposite to the one studied in Patient H. E. S.. arcs consistent with the proposition that transfer rat.es art: diminished in the Rh-sensitizrd state. Summary
Parameters of the transference of ampicillin among the three compartments I mother. fetus: and amniotic sac) have been studied in 103 patients. A rapid transfer occurs from the mother to the fetus, with fetal serum levels reaching levels exceeding rninimal inhibitory concentrations for many organisms causing intrauterine infections. The antibiotic appears in the amniotic fluid after 90 minutes; the amniotic fluid compartment appears to form a depot for, tht, sequestration of ampicillin. Varying ratrs of transfer out of the amniotic fluid arf’ found in different stages of pregnancy and pathologic conditions. The findina of a heat-labile system, inhibitory for the ,qrowth of Sarcirzo li~tczu, is recorded.
REFERENCES
Prylcs, C. V., Steg, N. L., Nair, S., Gellis, S. S.. and Tenney, B.: Pediatrics 31: 608, 1963. Charles, D., and Jacoby, H. E.: .4~. J. OBST. & GYNEC. In press, 1966. Ferrata, A.: Berlin klin. Wschr. 44: 366, 1907. Walsh, H., Hildebrandt, R. J., and Prystowsky, H.: Ahf. J. OBST. & GYNEC. 93: 590, 5. 6. 7. 8. 9. to. Il.
12. 13. 14. 15.
Kunin, C. M.: Proc. Sot. Exper. Biol. B Med. 117: 69, 1964. Charles, D.: J. Obst. & Gynaec. Brit. Emp. 61: 750, 1954. Bunn. P., Knight, R., and Amberg. J.: New York J. Med. 60: 3074, 1960. Prigot, A., Froix, C. J.. and Rubin, E.: Anti-
1965.
microb.
Elliott. R. B., Stokes, E. ,J., and Maxwell, G. M.: Arch. Dis. Childhood 39: 101, 1964. Belcher. T. E., Edgar, W. M., Melville, H. A. H., and Peel, K. R.: Brit. M. J. 1: 137: 1966. Rolinson, G. N.. and Stevens, S.: Brit. M. J. 2: 191, 1961. Quinn. E. L., and Cox, F.: Antimicrob. Agents & Chemotherap., 1963, p. 639. Eickhoff, T. C., Kislak, J. W., and Finland, M.: ,4m. J. M. SC. 249: 183, 1965. Weinstein. L.. Lerner. P. I.. and Chew. W. H.: New ‘E&land J. ‘Med. ‘271: 525, i964. Odell. G. B.: J. Pediat. 55: 268, 1959.
402. Kline, A. H., Blattner, R. J., and Lunin, 1. A. M. il. 188: 170. 1964. kohlan, S. Q., Beveiander, G., and Broz. i\ntirnicrob. Agents Pr Chemotherap.. p. 340. Conway, N., and Birt, B. D.: Brit. M. 260. 1965. Lisdhner, H., Seligman, S. T.. Krammer, and Parmelee. A. H.: 1. Pediat. 59: 21. Barber, M., Bnd Waterworth, P. M.: M. J. 2: 344, 1964.
16. 17.
18. 19. 20.
Agents
& Chcmotherap..
1962, p. M.: S : 1961. J. 2: A.. 1961. Brit.