0099-2399/90/1603--0135/$02.00/0 JOURNAL OF ENDODONTICS Copyright 9 1990 by The American Association of Endodontists

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VOL. 16, NO. 3, MARCH 1990

Distribution, Metabolism, and Excretion of

[14C] Glutaraldehyde

Don M. Ranly, DDS, PhD, and Diane Horn, BS

10 #Ci of labeled glutaraldehyde ([~4C]l,5-glutaraldehyde, 10.5 mCi/mmol; New England Nuclear, Boston, MA). Each animal, previously anesthetized i.p. with nembutal, was infused with a total volume of 50 ul of isotope over the time span of 1 min. One group of rats was killed at each of the following postinfusion intervals: 5 min, 1 h, 24 h, and 3 days. The rats were anesthetized with ether, and the blood was collected in heparinized tubes and centrifuged at 2800 rpm (Beckman J2-21) to separate cellular and humoral fractions. The packed ceils contained approximately 8.8 • 10 9 cells per ml when counted on a hemocytometer. Aliquots of the cells and plasma were then prepared for counting by liquid scintillation spectroscopy using Ready Protein scintillation cocktail (Beckman, Fullerton, CA). All vials were counted for 5 min, and quenching effects were corrected by an external 137Cs standard using the H-number method of quench correction. Counts are reported as dpm per ml of packed ceils or ml of plasma.

Radiolabeled glutaraldehyde (GA) was infused into rats in order to determine its distribution between cellular and humoral fractions of the blood, its potential metabolism by RBC's, its rate of excretion, and the nature of its urinary products. This study demonstrated that [I*C]GA was distributed between the RBC's and plasma at a ratio greater than 1. Although the absolute counts of both fractions dropped 80% over 3 days, the percentage bound or incorporated increased over time. Despite the extensive uptake by RBC's, these cells were unable to metabolize GA to CO2. Urinary excretion of the radiolabel was rapid; the predominant form in the urine was less than 1 kDa in size. All evidence suggested that it was not native GA. We conclude that the RBC's can incorporate GA, but can not metabolize it completely to CO2. Nevertheless, much of the infused GA was rapidly converted to nonreactive metabolites and eliminated by the kidney.

Dialysis Experiments Using Blood Cells and Plasma Two groups of rats (250 ___ 12 g) (four rats per group) were infused with 10 tsCi of radiolabeled GA (50 #1). One group was bled at 5 min and another at 1 h, and the cellular and humoral fractions of the blood were separated in the manner described above. One-milliliter samples of cells or plasma were then dialyzed against buffered saline, using Mr 50,000, 12,000, or 1,000 cut-off dialysis tubing (Spectrum, Los Angeles, CA). The retentate was then evaluated for radioactivity.

In recent years glutaraldehyde (GA) has been recommended as an alternative to formocresol for pulpotomy procedures in primary teeth (1). It has also been recommended as a disinfectant in root canal therapy (2). Glutaraldehyde has excellent fixative properties (3, 4), low antigenticity (5), shallow penetrating ability (6, 7), favorable pulp acceptance (7), and encouraging preliminary clinical results (8, 9). Miniscule amounts of GA have been shown to be distributed systemically following application to an amputated pulp, but the margin of safety for systemic glutaraldehyde has been shown to be very high (10). Because the introduction of any new drug into the profession today requires multiple preclinical investigations of efficacy, mode of action, metabolic disposition, and safety, this study was conducted to investigate some further aspects of the distribution, metabolism, and excretion of GA.

Comparative Studies of [14C]GAMetabolism by Blood Cells and Liver Tissue Blood was harvested from the renal aorta of four anesthetized rats with heparinized syringes and centrifuged at 2800 rpm to pack the blood cells. The plasma was then decanted, and the cells were washed three times with Hanks' balanced salt solution. The packed blood cells were then brought up to a 20% volume with Hanks' balanced salt solution, and 5 ml of this solution were placed into each of four flasks sealed with rubber sleeve stoppers with attached plastic wells. To each of the vials [~4C]GA (3.3 #Ci) was added. Methylbenzethonium hydroxide (Sigma, St. Louis, MO) was then deposited in the plastic wells. The flasks were then incubated at 37~ in a shaking water bath for 1 h.

M A T E R I A L S AND M E T H O D S Distribution of [~4C]Glutaraldehyde ([14C]GA)between Cells and Plasma of Blood Four groups of Sprague-Dawley rats (four rats per group) weighing 250 + 10 g were infused via the jugular vein with

135

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Journal of Endodontics

Ranly and Horn

For the study of liver metabolism, rats were anesthetized with ether and the ventral lobes of the liver were excised. The lobes were quickly sliced with a Stadie-Riggs tissue slicer into pieces weighing approximately 500 mg. The sections were then placed in preweighed flasks containing 2 ml of Hanks' balanced salt solution. For a control, two tissue slices were heated at 650C for 30 rain. After reweighing, the flasks were sealed with rubber sleeve stoppers with attached plastic wells. Methylbenzethonium hydroxide was added to the plastic wells, and 1.5 uCi of [14C]GA was added to the tissue. The sealed flasks were then shaken at 37~ in a water bath for lh. After the incubation of blood cells or liver slices, the reaction was stopped with NaOH. After 30 min the cups of methylbenzethonium hydroxide were removed and placed in scintillation vials for counting. The RBC's were then washed and counted for incorporated radiolabel. Studies on Urinary Excretion of

[14C]GA

In order to minimize the reaction time of filtered GA with urinary peptides or tract structures, a catheter was surgically placed into the bladder. Four 225 _+ 9 g rats were anesthetized with ether, and an abdominal incision was made to expose the bladder. A small opening was cut in the bladder to permit the intrusion of a polyethylene tube with a retentive lip formed by flaming. The tube was secured by ligatures and the abdominal opening was closed. The rats were placed in restraining cages until their urine became free of hemorrhage. At that time, the rats were removed, infused with [14C]GA (10 #Ci), gavaged with 5 ml of tap water, and returned to the restraining cages. Urine was collected in graduated tubed for six 1-h intervals. The volumes of the hourly urine outputs were recorded. Aliquots of the urine were taken for counting by liquid scintillation; the value reported was adjusted for volume and represents the mean total radioactivity excreted during each hour of collection. Another group of five rats (227 + 10 g) was infused with radiolabeled G A in the same manner, catheterized, and gavaged 3 days later. The value reported represents the total output of radioactvity over a 2-h period.

Investigations on the Urinary Metabolites of

['4C]GA

Aliquots of urine from the first hour collections of the experiment described above were dialyzed using Mr 1000 cutoff dialysis tubing. The total activity of the aliquots was known, permitting the retentates to be reported as percentages of the total dialyzed material. Samples of urine from the first hour collections of the experiment described above were analyzed by thin layer chromatography using benzene:methanol 45:8, vol/vol as the solvent system. After chromatography, the areas of the plate associated with pure GA and the remainder were separately scraped from the plate, eluted, and counted by liquid scintillation. As controls, the following combinations were chromatographed: [14C]GA alone, control urine and [14C]GA, and the combination of control urine, 2% cold GA, and [14C]GA, spotted in sequence on the plate. A group of four rats (232 _+ 8 g) was catheterized in the

manner described above. After the effects of the surgery had cleared, they were infused with 10 uCi of radiolabeled GA, gavaged, and placed in restraining cages. Two milliliters of urine were collected in tubes containing 1 ml of 1% bovine serum albumin (BSA) (Sigma) for 2 h. The tubes were then treated with trichloroacetic acid, allowed to sit overnight at 4~ centrifuged, and decanted. The supernatants were saved for counting. The BSA fractions were hydrolyzed with NaOH and aliquots were counted.

In Vitro Analysis of GA Cross-Linking to Humoral Proteins Aliquots of rat plasma (0.5 ml) were incubated with 25 #1 of [14C]GA (970, 848 dpm) for 5 rain, 1 h, and 24 h. At the end of incubation, 2 ml of 10% trichloroacetic acid were added to precipitate the proteins. The samples were then centrifuged at 3000 rpm for 10 min; the pellets were saved, washed, and centrifuged again. The pellets were solubilized with 3 M NaOH and counted by liquid scintillation spectroscopy. To investigate the cross-linking chemically, aliquots of serum (120 ul) were incubated with 10 ul of 2% G A for 5 min, 1 h, or 24 h. At the end of the incubation periods, SchilTs reagent was added directly to the samples, which were then analyzed at 540 n m after color development. Readings were adjusted for the absorbance of untreated serum, and the extent of the reaction of aldehyde moieties with Schiff's reagent is reported as a percentage of the total G A added.

Results Distribution Studies The distribution of the 14C label between the cells and plasma can be seen in Table 1. The level of label was higher in the cellular fraction of blood than in the plasma when compared on a volume basis. The cell to plasma ratio varied between 2 and 3, depending upon the period of observation. The extent of plasma trapping was not determined in this study, but because the radioactivity was compared on a volume basis, the higher values for the cellular fractions can only be ascribed to cell incorporation. Within 3 days, there was a 6-fold reduction in both the cellular and humoral label. Although the cells may have incorporated GA, it was eliminated at a rate proportional to that of plasma. The results of dialysis of the cellular and humoral fractions are reported in Table 2. There was a significant difference in the amount of radiolabel associated with the cellular elements as opposed to the portion bound to plasma. This held true for TABLE 1. Distribution of [14C]GA between cellular and humoral components (dpm/ml of plasma or cells) Time 5 min 1h 24 h 3 day

RBC 118,879 49,540 23,719 20,801

+ 23,171 * ___15,479 ___2,823 ___2,002

9 Mean _+ SD of samples from four rats per group.

Plasma 35,260 26,708 14,845 6,375

4- 4,186 _+ 5,988 _+ 4,094 4- 2,873

Glutaraldehyde Metabolism, and Excretion

Vol. 16, No. 3, March 1990 TABLE 2.

[14C]GAin retentate of plasma and

RBC fractions

5-min Postinfusion

Plasma RBC's

137

1-h Postinfusion

50,000

12,000

1,000

50,000

12,000

1,000

2,136 _ 410" 22,743 ___4,298

2,279 -+ 248 17,385 -+ 3,069

2,400 4- 197 19,339 _+ 3,963

3,947 _+ 1,618 27,801 _+ 17,271

4,542 _+ 2,060 23,798 -+ 16,598

3,959 - 858 21,698 4- 17,758

* Mean -+ SD, n = 4 at each time interval.

both time periods and for all pore sizes of tubing. The RBC to plasma ratio of the retentates ranged from 5 to 10 to 1, depending upon the postinfusion interval. Therefore, a comparison of the distribution of label among the two fractions previously described and the retentates in this experiment suggest that incorporation of the isotope into the cells was an important factor in establishing the cell to plasma ratio. The fact that the results were virtually identical regardless of the pore size of the tubing also suggests that the dialyzable labeled molecule was probably native GA or a similarly sized metabolite. When the data from Tables 1 and 2 are combined, the distribution between fractions and time periods can be better appreciated (Table 3). For these calculations, the counts used for the retentates were averages of the three molecular weight cut-off values; the decision to use the larger sample size was based on the fact that they were not different statistically. In the 5-min groups, approximately 5% of the radiolabel remained bound to plasma proteins after dialysis; approximately 16% remained associated with the ceils. In contrast, at 1 h, approximately 12% remained linked to the plasma proteins and 50% to the cells.

TABLE 3. Percentage of 14C radioactivity associated with retentate of dialyzed plasma and RBC's Time (min)

Plasma (%)

RBC's (%)

5 60

6.4 15.5

16.6 49.3

TABLE 4. Metabolism of

[14C]GAby RBC's and liver slices

in

vitro

RBC'st (dpm/ml RBC's) Intact (n = 4) Hemolyzed (n = 4) Liver (dpm/g tissue) Intact (n = 4) Heat-denatured (n = 2)

14CO2

Level of* Significance

1,806 _+ 1,129 2,175 + 276

NS

47,221 _ 7,245 2,665 _+ 115

p < 0.001

* S t u d e n t ' s t test. t O n e milliliter of paoked cells weighing 1.1 g.

TABLE 5. Urinary excretion of [14C]GA

Metabolism Studies From the data in Table 4, it can be concluded that the RBC's did not metabolize GA. The difference between the amount of'4CO2 produced by the intact versus the hemolyzed cells was not significant. In data not shown in Table 4, 20% of the isotopic load was found to be incorporated into the RBC's. The liver slices, on the other hand, converted GA to CO2. There was a 17-fold greater production of CO2 by viable liver cells than those killed by heat treatment. There was also a similar magnitude of difference between the intact RBC's and liver slices. Since each flask of blood cells contained 1 ml of packed RBC's (1.1 g per ml), almost direct comparisons can be made between the RBC's and slices on a weight basis.

Urinary Excretion The urinary excretion of [14C]GA is reported in Table 5. The rapid drop in blood [~4C]GA was reflected in the urinary output. During the first hour, approximately 14% of the infused radiolabel was excreted. The rate of excretion dropped over the next 2 h, after which it tended to level off over the succeeding 3 h. During the first 6 h, approximately 29% of the infused dose was eliminated. After 3 days the kidney was still active in removing '4C, even though the hourly output had dropped significantly.

Time First h Second h Third h Fourth h Fifth h Sixth h Third d a y t 2-h collection

dpm/Collection period* 3,015,173 _+ 1,069,962 1,311,424 _+ 621,997 722,871 _ 152,784 392,636 _+ 100,554 365,152 _+ 51,493 394,458 _+ 58,998 91,969 4- 18,648

% of Dose 14 6 3 2 2 2 0.2/h

* Mean -+ SD, n = 4 for t h e first 6-h collection. t M e a n -+ SD, n = 5 for the 2-h collection after 3 days.

Urinary Metabolites The results of urine dialysis reported in Table 6 demonstrated that the metabolites of GA, even though they were excreted either as the native molecule, as conjugated forms, or linked to amino acids or small peptides, were less than 1 kDa in size. Presumably, GA that became linked to blood constitutents such as cells or plasma proteins was degraded prior to glomerular filtration. When the urines were collected directly into BSA, precipitated with acid, and separated into fractions, the ~4C was preponderantly distributed in the supernatant (Table 6). Results of chromatography with pure labeled GA and urine are reported in Table 6. Ninety-three percent of the applied [14C]GA was eluted from the silica plate at the Rr of pure GA, whereas only 3% of the label in the urine migrated the same

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Ranly and Horn

Journal of Endodontics

TABLE 6. Analysis of 1'C in urine

Experiment* Dialysis of urine Retentate Diffusate Binding to BSA BSA Fraction Supernatant Recovery by thin layer chromatography Pure [14C]GA First h urine

% dpm

TABLE 7, Glutaraldehyde Cross-linking to plasma or serum (reported as % bound to protein)

Radiolabel 3.2 96.8 4.0 96.0 93.0 3.0

Length of Treatment*

Precipitated (%)

After Dialysis (%)

50 58 72

43 57

5 min 1h 24 h

Chemical (%) 40 72 80

* n = 4 f o r all t i m e p e r i o d s .

* n = 4 f o r all a s s a y s ,

distance. The extent of the binding of GA to serum proteins can be seen in Table 7. DISCUSSION The studies (1, 7) which support GA as an alternative to formocresol for pulpotomies have prompted research related to its systemic distribution, metabolism, and toxicity (10, 11). This study was undertaken to further investigate its metabolism and elimination, and, indirectly, pathogenic potential. The results of our distribution study agree with the findings of Myers et al. (11) who reported that the ratio of RBC-[~4C] -GA to plasma-[14C]GA (RBC to plasma ratio) was greater than one following treatment of teeth. In addition, the results of the present study paralleled previous findings that showed the rapid fall of serum GA over time (10). Interestingly, the RBC to plasma ratio did not change dramatically over the 3day test period. The intent of the dialysis study of the cellular and humoral fractions was to investigate the extent of binding of ['4C]GA to plasma protein and RBC's, and to determine if there was a particular range of plasma protein that was associated with it. Accordingly, fractions of rat blood were dialyzed in tubing that varied in pore size; we hoped to determine the lability of the associations and the size of the [~4C]GA complexes, if any. It should be noted that even though the total amount of ~4C distributed between cells and plasma dropped between 5 min and 1 h (see Table 1), the amounts associated with the retentates did not fall in parallel (see Table 3). Instead, the absolute amount of plasma radiolabel in the retentate rose between 5 min and 1 h, suggesting that with time, GA became cross-linked to plasma proteins. There were no significant, absolute differences in the retention of '4C between 5-min and 1-h dialyzed cells, even though an overall reduction of approximately 60% was noted in the cellular radiolabel. These data suggest that despite an overall reduction in [14C]GA in plasma and cells, an increasing percentage of the remaining label associated into nondialyzable forms or was incorporated into cells, respectively. It has been demonstrated that GA can be metabolized to CO2 by mammalian tissue (12). Since RBC's incorporated the majority of the blood-borne ['*C]GA in the current study, the role of the RBC's in this process was investigated. Despite the large uptake of GA by the blood cells (20%) in the metabolic flasks, they were not able to metabolize GA to CO2. The liver slices, on the other hand, were readily able to convert GA to CO2. This activity was demonstrated previously by Karp et

at. (12) and the experiment was included in this study as a verification of our culture system. Two liver slices were heat denatured as an additional control measure. Because two laboratories (10, 11) have demonstrated that the kidney plays a major role in the elimination of GA, we wanted to investigate the form or forms of urinary [~4C]GA as part of our studies on its metabolism. During the first 6 h, approximately 29% of the infused dose was eliminated. After 3 days the kidney was still active in removing J4C, even though the hourly output had dropped dramatically. In our previous study (10), almost 90% of the body load of [~4C]GA had been eliminated 3 days postinfusion. Decarboxylafion and conversion of GA to CO2 combined with active kidney filtration served to rapidly eliminate GA from the body of rats. Although it is obvious that ~4C is excreted as a large percentage of the administered load of radiolabeled GA, the exact form (or forms) of the urinary metabolite is unknown. Much of the radioactivity excreted immediately after infusion could be the native molecule, but our data suggest that this is n o t SO.

First, the collection of urine immediately into a solution of BSA demonstrated virtually no cross-linking molecules and, by inference, no native GA. This is not too surprising, since, considering the great reactivity of GA, one might expect binding to urinary tissues such as the bladder, if the GA were not already bound to excreted amino acids or peptides. If GA did bind to urinary proteins, the reaction products were not large, since virtually all of the radiolabel escaped from dialysis tubing with a Mr 1000 cut-off. Second, TLC data support our conclusion that native GA is not likely to be excreted in significant proportions. When pure [~4C]GA was chromatographed, approximately 93% of the label could be eluted from the spot which moved to the Rf of GA. When urine from the first hour of collection was chromatographed, less than 3% could be accounted for at this Rf. The remaining radioactivity remained at the origin or trailed behind the GA. When control urine was spotted on the chromatogram, and then overlaid with pure [t4C]GA, the eluate from the silica taken at the Rr of GA exhibited approximately 4% of the potential label. We conclude that the native molecule quickly reacted with constitutents of urine, which modified its movement on the chromatogram. One further control supports this conclusion. When control urine was spotted at the origin, followed by 2% GA, and then [14C]GA, 55% of the radiolabel could be eluted from the gel at the area corresponding to GA. We suggest that the cold GA neutralized many of the substrates of urine, lessening the cross-linking with ['4C]GA which was applied last. We conclude therefore, judging from the rapidity with which the radiolabel crosslinked to urine components on the silica plate, that it is

Vol. 16, No. 3, March 1990

unlikely that any native GA would exist in urine. We attempted to estimate the rapidity and stability of glutaraldehyde cross-linking to blood proteins by radiolabel and colorimetry in vitro (Table 7). Although the two methods gave somewhat different values, both demonstrated that glutaraldehyde is capable of reacting rapidly and permanently with the humoral components of blood. These results explain the binding of radiolabel to plasma in vivo, even after lengthy dialysis (Table 3). Our results suggest that GA is rapidly modified, perhaps oxidized to forms such as glutaric acid. Clearly, the level of radiolabel in the undialyzed plasma measured considerably more than in the retentate of the dialyzed samples. We conclude that the majority of the radiolabel which escaped from the dialysis tubing had already been converted to a noncrosslinking form. Of those molecules of GA which do become bound to blood proteins or cells, the majority must be eventually metabolized to CO2 or other small molecules. Clearly, the radiolabel is cleared from the body of rats, and virtually all of it is less than l kDa when excreted. The RBC's, which incorporate the majority of blood GA, may contribute to this process, even though they seem incapable of metabolizing the molecule completely to CO2. Our results further suggest that the precise identification of the blood and urinary metabolites of GA will be very difficult. There are likely to be many of them, since, besides direct modification of the native molecule, the cross-linked reaction products are eventually degraded. However, the precise determination of the metabolites may be unnecessary if our motivation for gaining the knowledge stems from a concern about the potential systemic toxicity of GA. In this regard, a recent study (l l) demonstrated the wide margin of safety in the use of GA as a pulpotomy agent. The present study helps explain the low toxicity of GA. Although a Xenbiotic molecule, it is

GIutaraldehyde Metabolism, and Excretion

139

neutralized by several factors: oxidation to CO2, urinary excretion, and cross-linking to plasma proteins. This study was supported by NIDR Grant DEO 7950-2. Dr. Ranly is professor, Pediatric Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX. Ms. Horn is research assistant, Pediatric Dentistry, University of Texas Health Science Center at San Antonio.

References 1. Davis MG, Myers R, Switkes MD. Glutaraldehyde: an alternative to formocresol for vital pulp therapy. J Dent Child 1982;49:176-80. 2. s'Gravenmade EJ. Some biochemical considerations of fixation in endodontics. J Endodon 1974;1:233-7. 3. Flitney FW. The time course of the fixation of albumin by formaldehyde, glutaraldehyde, acrolein and other higher aldehydes. J R Microsc Soc 1986;85:353-64. 4. Ranly DM, Hom D, Zislis T. The effect of alternatives to formocres~ on antigenicity of proteins. J Dent Res 1985;64:1225-8. 5. Ranly DM, Lazzari DP. A biochemical study of two bifunctional reagents as alternatives to formocresol. J Dent Res 1983;62:1054-7. 6. Ranly DM, Garcia-Godoy F, Horn D. Time, concentration, and pH parameters for the use of glutaraldehyde as a pulpotomy agent: an in vitro study. Pediatr Dent 1987;9:199-203. 7. Kopel HM, Bemick S, Zachrisson E, De Romero SA. The effects of glutaraldehyde on primary tissue following coronal amputation. An in vivo histologic study. J Dent Child 1980;47:425-30. 8. Wemes JC, Jansen HWB, PurdelI-Lewis D, Boering G. Histologic evaluation of the effect of formocresol and glutaraldehyde on the periapical tissues after endodontic treatment. Oral Surg 1982;54:329-32. 9. Garcia-Godoy F. A 42 month clinical evaluation of glutaraldehyde pulpotomies in primary teeth. J Pedod 1986;10:148-55. 10. Ranly DM, Horn D, Hubbard GB. Assessment of the systemic distribution and toxicity of glutaraldehyde as a pulpotomy agent. Pediatr Dent 1989; 11:8. 11. Myers DR, Pashley DH, Lake FT, Burnham D, Kalathoor S, Waters R. Systemic absorption of 14C-glutaraldehyde from glutaraldehyde-treated pulpotomy sites. Pediatr Dent 1986;8:134-8. 12. Karl:) WB, Korb P, Pashley DH. The oxidation of glutaraldehyde by rat tissues. Pediatr Dent 1987;9:301-3.

Distribution, metabolism, and excretion of [14C] glutaraldehyde.

Radiolabeled glutaraldehyde (GA) was infused into rats in order to determine its distribution between cellular and humoral fractions of the blood, its...
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