Bicarbonate-catalyzed hydrolysis of hexamethylene diisocyanate to 1,6-diaminohexane

ToxicologyLetters, 56 (1991) 1733178 0

1991 Elsevier Science Publishers

173

B.V. (Biomedical

Division)

0378-4274/91/S

3.50

ADONIS0378427491000633 TOXLET

02546

Bicarbonate-catalyzed hydrolysis of hexamethylene diisocyanate to 1,6-diaminohexane

Michtile Berodel, Bernard Testa and Heikki Savolainenl 1Institute qfOccupationa1 Health Sciences and 21n.Wute of Medicinal Chemi.str.v,University qf Lausanne Luusanne (Snkerland) (Received

30 August

(Revision

received 6 November

1990)

(Accepted

7 November

1990)

1990)

Kept Kurds; Hexamethylene

diisocyanate;

Hydrolysis;

I,6-Diaminohexane;

Bicarbonate;

Catalysis

SUMMARY The hydrolysis system. Without

of hcxamethylene catalysts,

diisocyanate

the reaction

carboxyhc-acid-containing

neutral

known

The catalytic

hydrolysis

acid and carbonic

product.

acid increased

at very high concentrations. tration

buffers markedly

occupational

catalyses

the formation

in this order while phosphate,

the rate of HDI hydrolysis following

in water was tested in a dynamic

and stationary

1%in IO min at 30°C) while the addition of simple of 1,6_diaminohexane

as the

efficiency of formic acid, oxalic acid, acetic acid, lactic acid, citric

A 20 mM bicarbonate

HDI in the lungs may be cataIysed metabolites

(HDI)

was very slow (<

was drastically by bicarbonate

glycine and glutamate

buffer was the optimal reduced.

It is suggested

in the blood,

catalyst,

were inactive

even

but below this concen-

that the hydrolysis

giving rise to amines

found

of inhaled as urinary

exposure.

INTRODUCTION

The reactivity of the isocyanate group serves as a basis for polymerization reactions to produce various polyurethanes. This reactivity may also explain toxic reactions seen in persons with frequent or repeated contacts with organic isocyanate monomers. However, the mechanism of the toxic reactions remains unclear. Allergy to isocyanate-modified macromolecules, is a frequently evoked hypothesis. Although seductive in its simplicity, it is not supported by convincing evidence. About 20% of patients with isocyanate-related pulmonary problems show immediate bronchial reactivity in short-term, low-level provocation tests to airborne isocyanate

Addrrss.fbr

c~r~e.~~o~de~ce~ M. Berode. IUMHT,

Rue du Bugnon

19, CH-1005,

Lausanne,

Switzerland.

174

monomers.

Furthermore,

no correlation

culating IgE immunoglobulins [I]. Biological monitoring of isocyanate sponding amines amine metabolites

exists between exposure

the hypersensitivity

is possible

by urinalysis

and cir-

of the corre-

generated from the absorbed isocyanate monomers [2, 31. The have a short biological half-life due to their rapid N-acetylation

in the body [4]. All cells lining the trachea and bronchi are covered with the so-called ‘unstirred’ water layer [5] so that reactions of inhaled isocyanates are expected to begin in the lungs at this first contact with body water. Following inhalation, no free isocyanate has ever been detected in body fluids or urine. However, evidence has been presented that methyl isocyanate could form carbamoylates with glutathione and in this form it could be transported to peripheral organs [6]. A shortcoming of this hypothesis is that there is very little reduced glutathione in the extracellular water space or plasma [7]. Furthermore, isocyanates are not known to be substrates for intracellular glutathione-S-transferases which catalyze the formation of thioethers from electrophilic metabolites [8]. This makes the reaction with intracellular thiols an unlikely possibility since isocyanates display reactivity towards functional groups with acidic protons, e.g. alcohols, amines, carboxyls and carboxylic acids, while thiols react in the physiological milieu preferentially by forming short-lived thiyl radicals [9]. Acids and bases catalyse the hydrolysis of isocyanates to the corresponding amines, a fact that is exploited in industrial hygiene practices to trap airborne isocyanate in liquid impingers [lo]. Animal organisms contain a major carbonic acid buffer system capable of maintaining the body fluid pH within a narrow range [I I], the circulating blood bicarbonate concentration being in the order of 20 mM. In view of this large carbonate pool, we wondered whether bicarbonate ions could catalyze the hydrolysis of an industrially important diisocyanate to its diamino product. This hypothesis is verified here, for the first time, and we bring quantitative details of the reaction, comparing the catalytic efficiency of bicarbonate with that of other carboxylic acids. MATERIALS

AND METHODS

Hexamethylene diisocyanate (HDI, Fluka, Switzerland) atmospheres were generated with the so-called ‘permeation method’ [ 121. Liquid HDI was put in a glass ampoule connected to silicone tubing. This permeation system was kept in an air bath of constant temperature (an empty reaction bottle in a water bath at 30°C). Dry nitrogen was blown into the flask at 100 ml/min. This primary stream loaded with HDI vapours was then diluted with dried air (I.63 l/min) and, after mixing, was led into an expansion vessel (about 2 I) equipped with a 6-port sampling manifold so that many samples could be collected simultaneously. Air sampling was performed by bubbling for 10 min at 1 l/min into two successive midget impingers, one containing 10 ml of the desired buffer mixture and the second containing 10 ml of Marcali solu-

175

tion (0.4 N HCl+0.4 HDA for analysis.

N acetic acid) to trap the unreacted The HDI concentration

HDI and transform

in the test atmosphere

obtained

it into with this

dynamic technique could be regulated between 200 and 400 pg HDI/m3. The following buffers at pH 7.4 were examined for their capacity to catalyse

HDI

hydrolysis: phosphate, 0.2 and 0.05 M; glycine, 0.2 M in 0.2 and 0.05 M phosphate; glutamate, 0.2 M in 0.2 and 0.05 M phosphate; Krebs’ solution (20 mM NaHC03, 120 mM NaCl, 20 mM NaHzPOd, 1.3 mM CaC12 and 1.7 mM glucose). To evaluate the effect of the carbonate concentration on the hydrolysis yield, the NaHC03 content of the Krebs’ solution was varied between 20 mM and 20 PM. In a second set of experiments, the rate of HDI hydrolysis was investigated in a closed static system. One ml of HDI solution (50 ,ug/ml in acetone) was added to 10 ml of buffered solutions (pH 7.4) which contained 20 mM acetic, formic, oxalic, lactic or citric acid instead of 20 mM bicarbonate in Krebs’ solution. The mixtures were incubated at room temperature for 5-60 min and analyzed for 1,6-diaminohexane formed [IO] with 1,7_diaminoheptane as an internal standard. The results were compared wit those obtained in Krebs’ solution containing 20 mM bicarbonate. After hydrolysis of HDI to its derivative amine, an aliquot (2-10 ml) of the tested buffer solution was transferred to a test tube where appropriate volumes of 10 M NaOH (2-10 ml) and toluene (l-5 ml) were added. The internal standard was added (1,7_diaminoheptane) and the mixture was then shaken for 15 min at room temperature. The organic phase was separated by centrifugation and an aliquot was transferred to another tube where 30 ,ul heptafluorobutyric anhydride (HFBA, Supelco, U.S.A.) were added. The tubes were capped and transferred to a heating block warmed to 55°C for 1 h. The samples were then cooled to room temperature and shaken with 1 ml of 0.4 N NaOH to remove the excess of HFBA. The toluene layer containing the amides formed was then separated for chromatographic analysis. One microlitre was injected into a capillary gas chromatograph (Perkin-Elmer, Sigma 2B) equipped with an electron capture detector in the split mode. The separation was performed on a fused silica capillary column (25 m x 0.32 mm) with chemically bonded stationary phase, CP-SIL 5 CB (Chrompack, Middelburg, The Netherlands). RESULTS

Bubbling

of HDI vapour

in the reaction

vessel containing

2 x lop2 M bicarbonate

led to total hydrolysis of the HDI to 1,6-diaminohexane (Table I). When the bicarbonate concentration was reduced to 2 x 10e3 M, only 15% of the HDI was hydrolysed to the diamine, and below 2 x 1O-4 M bicarbonate practically all HDI vapour crossed the reaction impinger without reacting (Table I). This was also the case in the presence of phosphate, glycine and glutamate buffers (Table I). In the second reaction system, bicarbonate and citric acid buffers at each time point displayed the greatest catalytic efficiency (Fig. 1). It should be noted that the

176

TABLE

I

HYDROLYSIS

OF

HEXAMETHYLENE

BUFFER SOLUTIONS ____.___ .._._~

(pH 7.4,3O”C.

DIISOCYANATE

TO

1,6-DIAMINOHEXANE

IN

10 mitt) ___..__~__

Solution

Concentration

Hydrolysis

(Ml

n*

(99 _--..._.._..

~_._._

Water

_

Phosphate

2.10-I

<

5.10-’ Glycine

2.10-I

Gfutamate

2.10-I

Bicarbonate

2.10-’ 2.10

<:

----

1

2

2+0.5

3

17

1

1

2 3

6iO.h 94.7* 7

1.6

5

1 I I

13.6

2.10-”

5.9

2. IO-5

4.7

_--.-___

---.-I~

.~_

_.

*n, number of experiments. The reaction vapour captured

impinger

(200 gg/rn’)

contained

the solutions

was bubbled

in a second impinger

through

containing

at pH 7.4 at 30,‘C and a Bow of hexamethylene

it at a flow rate of the Marcaii

diisocyanate

1 hmin for 10 min. The escaping gas was

solution.

pH in the reaction vessels was close to that in the blood, sugesting that catalysis may be mediated by carboxylate anions. However, the less acidic catalysts (pK, > 6) were clearly much more effective than the more acidic ones (pK, < 5).

Acids tested [pK;J _

Lade

[3.0X]

Formic [3 751

IWDAl/IISI 3.5

--b

Oxalic [4 191

7_

Affiicj4.75]

Time [min] Fig, 1. Hydrolysis

of hexamethylene

diisocyanate

(HDI) in water at room temperature

to I,6-diaminohex-

ane (HDA) by various carboxylic acids 20 mM (pf-1 7.4). One ml HDI in acetone was added to 10 ml of each buffer solution. Aliquots were taken after 1, 5, 10, 20 and 60 min for HDA analysis using 1.7diaminoheptane

as internal

standard

(IS). The results are given as a ratio of HDA/IS.

177

DISCUSSION

The results of dynamic experiments (Table I) show that high proton concentrations are not needed for rapid and complete hydrolysis. Krebs’ buffer contains all the major inorganic constituents of blood, and its bicarbonate concentration (20 mM) corresponds to that of a normocapnic person [l 11.This fact strongly suggests that inhaled HDI may be quickly hydrolysed in the lungs, yielding 1,6-diaminohexane which is seen as a urinary metabolite of HDI [3]. Many authors have stressed the toxicological significance of tissue macromolecules reacting with organic isocyanates. For instance, to produce methylene diphenyl diisocyanate albumin complexes, purified serum albumin must be incubated with the diisocyanate in high concentrations for 24 h [13] and it should be remembered that this reaction is competed by hydrolysis in the lung. This reaction could still be a plausible mechanism for skin contact reactions although it would be less likely than in the lung where there is easy interaction with blood bicarbonate. The experiments with carboxylic acids other than carbonate indicate that the dissociation constant of the acids may be related to their reactivity. In this respect, the optimum pK, may be near 6 as the stronger acids (pK, < 5) were poor catalysts. Amino acids are inactive, perhaps because they exist as zwitterions in neutral media. Although the organic acids tested here have theoretical interest to help understand the reaction mechanism(s), only bicarbonate is of toxicological importance in its physiological concentration. Apart from this physiological importance, bicarbonate could also have applications in the technical prevention of HDI exposure. Baker’s soda is cheap and it could be used in scrubbers to purify the contaminated atmospheres. This compound is also safe and strong acids or strong alkali are not needed, and the waste would be dilute aqueous solutions of diaminohexane. In conclusion, bicarbonate very efficiently catalyzes the hydrolysis of HDI in water to its corresponding amine. This reaction is likely to be more effective than reactions with tissue macromolecules or glutathione in lungs, and it also explains the formation of N-acetyl- 1,6-diaminohexane as a urinary metabolite of HDI. ACKNOWLEDGEMENTS

We thank Mr. P.-A. Porchet for his excellent technical assistance and Professor M. Guillemin for his ideas and advice.

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