THAM and metabolic rate in the newborn guinea pig

THAM and metabolic rate in the newborn guinea pig MOLLY E. TOWELL, M.B., M.R.C.O.G., F.R.C.S.(C)* GIORGIO PARDI, M.D.** KARLIS ADAMSONS, M.D., PH.D. New York, New York The metabolic effects of tris-hydroxymethylaminomethane (THAM) in the newborn were investigated in neonatal guinea pigs. In 32 healthy piglets administration of THAM at pH 7.4 intraperitoneally in a dose of 1,200 mg. per kilogram did not affect oxygen consumption or the ability to maintain body temperature during exposure to cold. Furthermore the same dose of neutral THAM administered by slow intra-arterial infusion had no effect on the oxygen consumption of 12 anesthetized piglets. On the other hand, slow intra-arterial infusion of alkaline THAM or sodium bicarbonate at pH 8.6 led to a temporary increase in oxygen consumption. Rapid intra-arterial infusion of the alkaline solutions resulted in a profound though transient depression of respiration and oxygen consumption. It is concluded that THAM in the dose employed in these experiments does not appear to interfere with metabolic rate in the healthy neonatal guinea pig. The temporary alterations in ventilation and oxygen consumption observed following intravascular administration are due to pH of the infusate rather than to specific interference with metabolic processes.

carbonyl compound with TRAM. 1 It is not known whether this contributes to the hypoglycemic effect produced by administration of TRAM to human subjects or animals.2• 3 Depression of metabolic processes in the intact subject is reflected by reduction of total body oxygen consumption and by alteration of body temperature. Adult dogs under apneic oxygenation showed no change in oxygen uptake during administration of TRAM. 4 In the newborn monkey, on the other hand, slight reduction of oxygen consumption has been observed following the use of TRAM to correct acidosis due to asphyxia. 5 Although no adverse metabolic effects have been reported from the use of TRAM in the newborn infant, studies of toxicity have been confined to the adult. It is known that the effect of a pharmacologic agent is not necessarily the same in the newborn

THE AM IN E BUFFER tris-hydroxymethylaminomethane (TRAM) is used to correct respiratory and metabolic acidosis of the newborn infant. In vitro, TRAM interferes with certain enzyme systems by reacting with the substrate or with the enzyme. For example, the addition of TRAM to an aqueous solution of pyruvate leads to a decrease of the concentration of free pyruvate, probably due to the formation of a From the Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University. Supported by United States Public Health Grants HD 11802 and GM 09069. *Holder of Queen Elizabeth II Canadian Research Fellowship. Present address: Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia, Canada. **Fellow, Ministry of Public Education, Italy.

77

78 Towe II, Pardi, and Adamsons

May 1, 1970 Amer. J, Obstet. Gynec.

infant as in the adult. This investigation was therefore carried out to determine whf:ther TRAM, at a neutral pH and in doses recommended for correction of aci. dosis, could interfere with metabolic pro. cesses in the healthy newborn guinea pig. Material and methods TRAM (O.SM) was prepared with sodi· urn and potassium chloride in a 5 per cent dextrose solution and adjusted to a pH of 8.6 or 7.4 by addition of hydrochloric acid. This was done to avoid the adverse effects of a strongly alkaline solution. The solution contained 60 Gm. per liter TRAM, 50 mEq. per liter Na, and 8 mEq. per liter K; osmolality ranged between 1,000 and 1,350 mOsm. per kilogram depending on the pH of the solution. The dose of THAM administered to the piglet was 1,200 mg. per kilogram of body weight. A solution of urea with sodium and potassium chloride in 5 per cent dextrose was prepared for administration to control piglets. It contained 70 Gm. per liter urea, 50 mEq. per liter l'i'a, and 8 mEq. per liter K; osmolality was 1,500 mOsm. per kilogram. The dose of urea administered to the piglet was 1,400 mg. per kilogram of body weight. This composition was chosen to provide a nontoxic solution of similar molecular weight to match the osmolality of the THAM solution.

Solutions were administered in a volume of 20 mi. per kilogram of body weight by the intraperitoneal or intra.arterial route. Oxygen consumption was measured with a closed-circuit apparatus as illustrated in Fig. 1. Environmental temperature was maintained constant by a water bath in which the entire apparatus, except for the spirometer, was submerged. A How of air was created by a magnetically driven turbine pump. During observations the valve between the spirometer and the closed circuit remained open. Carbon dioxide was absorbed by soda lime, thus reduction in volume of the closed circuit due to consumption of oxygen by the animal resulted in movement of the spirometer which was recorded on a kymograph. The spirometer was refilled with oxygen evety 5 minutes. The colonic temperature of the piglet and that of the water bath and air in the animal chamber were continuously monitored with thermistors. Measurements were continued for at least 30 minutes after thermal equilibration had been reached within the closed circuit. Intraperitoneal injection in unanesthetized piglets. Sixteen pairs of newborn piglets aged 1 to 14 days and weighing 69 to 139 grams were closely matched for age and weight, and were divided into 2 groups, one receiving TRAM and the other urea. Oxygen consumption and body temperature of the

Spirometer

Kymograph

Fig. 1. Closed-circuit apparatus for measuring oxygen consumption.

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piglets were measured during exposure to a neutral thermal environment (water bath 32° C.) and a cold environment (water bath :20° C.) . The piglet was returned to its mother for one to 2 hours prior to intraperitoneal injection of THAM at pH 7.4 or urea. Immediately thereafter oxygen consumption was measured again at the same environmental temperatures. The sequence of exposure was varied; half of the piglets were first exposed to a neutral thermal environment and half were first exposed to a cold environment. Observations were completed within 2 hours following intraperitoneal injection. Intra-arterial injection in anesthetized piglets. Twelve newborn guinea pigs aged 1 to 11 days were anesthetized with Fluothane or secobarbital (10 mg. per kilogram intravenously) and the carotid artery was cannulated using a polyethylene catheter. The piglet was placed in the chamber of the closed circuit apparatus and oxygen consumption was measured before and after administration of THAM at pH 7.4 or 8.6 and 0.3M NaHC0 3 at pH 8.6. Water bath temperature was varied between 24° C. and 3 7° C. Intra-arterial infusion was carried out with a pump at a rate of 2.5 ml. per minute or 0.5 ml. per minute at intervals of not less than one hour, and only after a steady state of oxygen consumption and body temperature had been observed over a period of 15 to 25 minutes. Oxygr~n consumption was measured continuously for a period of 24 to 30 minutes thereafter. Six guinea pigs aged 2 to 30 days were anesthetized with secobarbital and were attached through a tracheotomy tube to the closed circuit apparatus to record oxygen consumption and respiratory rate.

Res.ults Effrects of intraperitoneal THAM and urea on unanesthetized animals. Administration of THAM at pH 7.4 or urea did not affect oxygen consumption in either a neutral thermal or a cold environment (Table I) . This was evident when comparison was

Metabolic effects on THAM 79

made of the means of the two groups and when each group was used as its own control. Ability to maintain body temperature was compared by measuring the fall of colonic temperature which occurred during the first 30 minutes of cold exposure. Administration of THAM at pH 7.4 or urea did not influence thermal stability (Table II). Effects of intra-arterial THAM and NaHC0 3 on anesthetized animals. Administration of THAM at pH 7.4 or 8.6 or NaHC0 3 at pH 8.6 by slow infusion (0.5 ml. per minute) did not appreciably change the total oxygen consumption during the 30 minute observation period. However, there was a marked increase in oxygen consumption during the initial 6 minutes after infusion of the alkaline solution of THAM or

Table I. Oxygen consumption in milliliters per kilogram per minute at STP (mean± S.E.) of newborn guinea pigs in a warm (32° C.) and cold (20° C.) environment before and after intraperitoneal administration of urea or THAM at pH

7.4 Thermal environment Warm Cold

Agent Vo. Before Vo. After Urea 17.6 ± 0.448 18.1 ± 0.687 THAM 18.0 ± 0.453 18.2 ± 0.584 Urea 37.7 ± 1.194 37.2 ± 1.137 THAM 38.8 ± 1.273 36.1 ± 1.399

Table II. Initial colonic temperature and its change during 30 minutes of cold exposure (L.it/30') in °C. (means± S.E.) of newborn guinea pigs before and after intraperitoneal administration of urea or THAM at pH 7.4

I

I

Agent Before Initial co- Urea 39.2 ± 0.104 Ionic tern- THAM 39.1 ± 0.129 perature At/30'

After 38.8 ± 0.238 38.6 ± 0.166

Urea -1.17 ± 0.164 -1.00 ± 0.188 THAM -0.74 ± 0.106 -0.96 ±

80 Towe! I, Pardi, cmd Adamsons

Amcr.

bicarbonate. This phenomenon was absent following administration of neutral TRAM (Table III). In contrast, rapid intra-arterial infusion (2.5 mi. per minute) of alkaline TRAM or bicarbonate was followed by marked depression of ventilation and oxygen consump-

J.

May 1, 1970 Obstet. Gyncc.

tion. Fig. 1 shows the effect of TRAM m, an anesthetized guinea pig; similar effects were observed following bicarbonate. Recovery was more rapid with bicarbonate administration and was followed by a prolonged increase in oxygen consumption (Fig. 2).

Table III. Change in oxygen consumption in per cent (mean and range) of anesthetized newborn guinea pigs at environmental temperatures of 24 to 37° C. after slow intra-arterial infusion of THAM at pH 7.4 or 8.6 and N aHC0 3 at pH 8.6 Time after infusion

pH

No.

0 to 6 minutes

6 to 12 minutes

THAM

7.4

12

THAM

8.6

8

NaHCO,

8.6

9

+1.7 (-4.0 to + 5.6) + 19.0 (+ 8.3 to + 58.1) + 13.7 (- 6.0 to + 31.7)

- 3.2 (- 8.7 to+ 4.0) + 2.8 (+ 3.9 to+ 10.6) + 8.5 (- 2.0 to + 30.4)

ml 02

riNFtiSION OF THAMl

20[ 10 0 0

3

Minutes

6

9

ml 02

Minutes

12

9

~:r.,, ~ 0[ ... ~ ?.'...

15

Minutes

18

Fig. 2. The effect of rapid intra-arterial injection of 0.5M THAM (20 mi. per kilogram) at pH 8.6 upon O, consumption and ventilation of a young guinea pig. Spirometer tracing showing frequency of respiration and tidal volume; the vertical line at the end of each 3 minute interval indicates milliliters of 0, consumed.

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Metabolic effects on THAM

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Comment

The effect of a neutral solution of THAM administered by the intraperitoneal route on oxygen consumption and maintenance of body temperature in newborn guinea pigs was smdied in a neutral thermal and a cold environment. This enabled the effect of THAM to be observed under conditions in which metabolic rate was minimal as well as close to maximal. It was anticipated that any :interference with metabolic processes not apparent under basal conditions would be revealed by exposure to thermal conditions which increase oxygen uptake. Total body oxygen consumption in both warm and cold environments and the maintenance of body temperature on exposure to cold were not statistically different after intraperitoneal administration of THAM, nor was there a difference between the effect of THAM and urea of similar osmolar concentration. Thus it is concluded that neutral THAM in the dose employed in these experiments had no effect on total body oxygen consumption or thermoregulation in the newborn guinea pig. A neutral solution of THAM (pH 7.4) was used to avoid the undesirable effects of strongly alkaline solutions administered to nonacidotic subjects. Neutralization of 0.5M THAM by addition of hydrochloric acid increases the osmolar concentration of the solution to 1,300 mOsm. per kilogram. Since it was not known whether such a hyperosmolar solution might itself cause an adverse effect on metabolic rate, a solution of similar osmolality was prepared for administration to control piglets; the choice of urea for this purpose depended on its properties of nontoxicity and free diffusion across cell membranes. It is of interest that solutions of such high osmolality could be administered to the newborn piglet without apparent effect on metabolic rate. Ionization of THAM increases as pH of the solution is reduced by addition of acid. At pH 10.2 only about 1 per cent exists in the ionized form but at pH 7.4 about 70 per cent is present as THAM-H+. Increasing ionization renders. THAM less effective as a buffer and may reduce free diffusion across

81

cell membranes; it does not, however, appear to reduce acute toxicity of the agent. 6 When an alkaline solution of THAM is administered to correct acidosis, conversion to the ionized from occurs as hydrogen ions are accepted. Although rapid penetration of the cell by nonionized THAM has been cited as one of its chief advantages in correction of intracellular acidosis, equilibration between intracellular and extracellular compartments may not be complete until 4 to 6 hours have elapsed. 7 Thus, interference with intracellular metabolic processes might not be immediately apparent following administration of THAM. In the neonatal guinea pig no difference was observed when intraperitoneal THAM was compared with urea even though measurements of oxygen consumption were continued for as long as 2 hours following intraperitoneal injection. The absorption of THAM from the peritoneal cavity is probably more rapid than formerly thought, when the use of this agent was proposed to act as an H-ion acceptor for weak acids in peritoneal dialysis. 8 • 9 Using C 14 -tagged THAM, Nahas and associates10 have shown that about 40 per cent of THAM is absorbed within 30 minutes from a peritoneal dialysate solution with an initial pH value of 9.8. Exposure to a large surface area and a wide gradient of concentration from peritoneal cavity to circulating plasma should favor rapid and efficient absorption. However, high osmolality of the solution employed in the neonatal

ml 02

NaHC03

~ vvVV1!Vl 0

12

24

36

Minutes Fig. 3. The effect of rapid intra-arterial injection of 0.3M NaHCOa (20 mi. per kilogram) at pH 8.6 upon 0, consumption of a newbbom guinea pig.

May!, 1970

82 Towell, Pardi, and Adomsons

guinea pig may have delayed transfer across the peritoneal membrane owing to the dilution of intraperitoneal contents which occurred following injection. Since the dose of THAM employed in these experiments ( 1,200 mg. per kilogram) was more than twice that recommended as the maximum single: dose in human subjects, 11 it was anticipated that any adverse effects might be apparent even if absorption was incomplete. Since the amount of TRAM absorbed from the peritoneal cavity was not precisely known, the effects of a similar dose administered by intra-arterial infusion were also studied. Although slow infusion of both neutral (pH 7. 4) and alkaline (pH 8.6) TRAM had little effect on mean oxygen consumption over a period of 30 minutes after infusion, alkaline TRAM (pH 8.6) led to a temporary increase of oxygen uptake in the first 6 minutes. A similar effect was observed following intra-arterial infusion of an alkaline mixture of sodium bicarbonatecarbonate (pH 8.6). Thus the alkalinity of the solution rather than the agent itself appeared to be responsible for the increase in oxygen consumption. A similar effect was observed after infusion of 0.5 TRAM at pH 8.8 to correct acidosis during resuscitation of the asphyxiated newborn monkey. 5

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J. Obstet. Gynec.

Although a fall in C0 2 tension in alveolar gas and tissue fluid following alkalinization of blood will increase the 0 2 space within the system, it does not appear to be sufficient magnitude to account for the observed differences in oxygen consumption. Fast intra-arterial infusion of alkaline TRAM and bicarbonate in the healthy anesthetized guinea pig caused profound depression of respiration and oxygen uptake in the first 5 to 10 minutes after infusion. It is not certain whether a similar effect would be observed after infusion of alkaline TRAM to correct acidosis. However, these results suggest that rapid infusion of an alkaline solution may be hazardous. Conclusion

It is concluded that TRAM in the dose employed in these experiments does not interfere with oxygen consumption of the healthy neonatal guinea pig. The temporary alterations in ventilation and oxygen consumption observed following intravascular administration are due to the alkaline nature of the infusate rather than to a specific interference of the TRAM molecule with metabolic processes. The authors wish to express their gratitude to Mrs. Christina Takle for skilled technical assistance.

REFERENCES

1. Mahler, H. R.: Ann. N. Y. Acad. Sci. 92: 426, 1961. 2. Bennett, T. E., and Tarail, R.: Ann. N. Y. Acad. Sci. 92: 651, 1961. 3. Tarail, R., and Bennett, T. E.: Proc. Soc. Exp. Bioi. Med. 102: 208, 1959. 4. Ligou, J. C., Le Tallec, Y., Bernadet, P., Broue, A., and Calazel, P.: Ann. N. Y. Acad. Sci. 92: 617, 1961. 5. Adamsons, K., Jr., Behrman, R., Dawes, G. S., James, L. S., and Koford, C.: J. Pediat. 65.: 807, 1964. 6. Roberts, M., and Linn, S.: Ann. N. Y. Acad. Sci. 92: 724, 1961.

7. Holmdahl, M. H., and Nahas, G. G.: Amer. J. Physiol. 202: 1011, 1962. 8. Knochel, J. P., Clayton, L. E., Smith, W. L., and Barry, K. G.: J. Lab. Clin. Med. 64: 257, 1964. 9. Sanger, C., Nahas, G. G., Goldberg, A. R., and D'Allessio, G. M.: Ann. N. Y. Acad. Sci. 92: 710, 1961. 10. Nahas, G. G., Gjessing, J. J., Giroux, J. ]., Verosky, M., and Mark, L. C.: Clin. Pharmacal. Therap. 6: 560, 1965. 11. Nahas, G. G.: Clin. Pharmacol. Therap. 4: 784, 1963.