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Ada Anaesthesiol Scand 1991: 35: 541-544 The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base? L. HEYTENS', J. J. A. HEFFRON~ and F. CAMU~ 'Department of Intensive Care, University Hospital Antwerp, Antwerp, Belgium, 'Department of Biochemistry, University College, Natioiral IJniversity of Ireland, Cork, Ireland and 3Department of Anaesthesia, Vrije Universiteit Brussel, Brussels, Belgium The aim of the present study was to investigate whether the three different caffeine preparations - caffeine citrate, caffeine benzoate and the free base - used for in uilro diagnosis of malignant hyperthermia susceptibility - produced the same amount of contracture in rat diaphragm. At equirnolar caffeine concentrations, thr pure base generated more tension in the rat diaphragm muscle than caffeine benzoate or caffeine citrate. The citrate lowers the pH and the free Ca2+ concentration of the test bath and thus suppresses the caffeine contracture. The benzoate is believed to inhibit the caffeine contracture by its carbonyl group in a way similar to the effect of benzocaine. Received I0 July 1990, accepted for publication 9 January 1991 Key words: Caffeine; contracture; malignant hyperthermia; muscle. Thr in vitro caffeine contracture test has a well-estab- lishcd value in the laboratory diagnosis of malignant hyperthermia (MH). It is either used as one single test (I), as a combined halothane-caffeine contrac- turr test (2) or as a combined test in which muscle tissue is exposed separately to caffeine and to halo- thane (3-5). Muscle from individuals susceptible to MH shows an increased in vitro sensitivity to halo- thane and caffeine compared to normal controls. Ac- cording to the European Malignant Hyperthermia Group's protocol (5), the diagnosis of MHS requires an increase in baseline tension of at least 0.2 g at 2% halothane or less and 2 mmol/l caffeine or less. Even wit 11 this standardised protocol, further specifications seem necessary. Kwent meetings of the European Malignant Hyperthermia Group have shown the need to mrasure the actual halothane concentration in the tissue bath rather than to depend blindly on the no- minal halothane concentration of the vaporizer (6). Also, because caffeine exists as the pure base or the morr soluble citrate or benzoate complexes, it sermed worthwhile to investigate whether these three preparations produce the same amount of contrac- ture when used in equal caffeine concentrations. Be- cause the number of muscle strips necessary for this experiment is difficult to obtain during muscle bi- opsy in humans, a rat model was used. MATERIAL AND METHODS Three groups of five rats were studied. Male Wistar rats weighing 250 to 400 g were anaesthetized with nembutal injected into the peritoneal cavity. The left upper quadrant of the diaphragm was dissected and four strips (2-3 mm wide and 15-25 mm long) werr taken from each rat. The muscle strips were then mounted in the tissue bath, which was continuously perfused with Krebs-Ringer solution at 37°C and carboxygenated (5). One end of the muscle strip was clamped rigidly and the other attached to a strain gauge connected to an amplifier/ recorder system. The muscle strip was stretched to 2 g and left for 15 min to stabilise. The muscle specimens were electrically stimulated by 1 ms supramaximal stimuli at a frequency of 0.2 Hz to prove viability. After obtaining a stable baseline, the bath was perfused with increasing concentrations - I, 2, 4 and 6 mmol . I-' of caffeine citrate, caffeine benzoate or caffeine pure base. Each concentration was maintained for at least 3 min or until a plateau of maximal tension was obtained. The caffeine preparations used were: caffeine, free base, biochem- ical grade (97.4% pure when assayed spectrophotometrically at 274 nm in ethanol) (from BDH Chemicals Ltd., Poole, Dorset, England); caffeine citrate, B.P. grade (49.3 to 50.4O6 caffeine) (from Allied Pharmaceutical Distributors, Dublin, Ireland), and caffeine benzo- ate, sodium salt, (from Sigma Chemical Co., St. Louis, MO, U.S.A.). Both pH and ionized Ca'' were measured at the different conren- trations of the three caffeine preparations in the Krebs solution. pH was measured directly in the tissue bath using a Radiometer PHM82 meter with a combination electrode. Ionized Ca2' was measured ar 37°C using a Radiometer F2112 calcium electrode and PHM62 researrh meter. The electrode was calibrated with standard calcium solutions in the range 0.1 to 10 mmol.l-'. Statistical analysis of the results was carried out using a paired Student &test; differences were judged significant when P was < 0.05.

The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base?

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Page 1: The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base?

Ada Anaesthesiol Scand 1991: 35: 541-544

The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base? L. HEYTENS', J. J. A. HEFFRON~ and F. CAMU~ 'Department of Intensive Care, University Hospital Antwerp, Antwerp, Belgium, 'Department of Biochemistry, University College, Natioiral IJniversity of Ireland, Cork, Ireland and 3Department of Anaesthesia, Vrije Universiteit Brussel, Brussels, Belgium

The aim of the present study was to investigate whether the three different caffeine preparations - caffeine citrate, caffeine benzoate and the free base - used for in uilro diagnosis of malignant hyperthermia susceptibility - produced the same amount of contracture in rat diaphragm. At equirnolar caffeine concentrations, thr pure base generated more tension in the rat diaphragm muscle than caffeine benzoate or caffeine citrate. The citrate lowers the pH and the free Ca2+ concentration of the test bath and thus suppresses the caffeine contracture. The benzoate is believed to inhibit the caffeine contracture by its carbonyl group in a way similar to the effect of benzocaine.

Received I0 July 1990, accepted for publication 9 January 1991

Key words: Caffeine; contracture; malignant hyperthermia; muscle.

Thr in vitro caffeine contracture test has a well-estab- lishcd value in the laboratory diagnosis of malignant hyperthermia (MH). It is either used as one single test ( I ) , as a combined halothane-caffeine contrac- turr test (2) or as a combined test in which muscle tissue is exposed separately to caffeine and to halo- thane (3-5). Muscle from individuals susceptible to MH shows an increased in vitro sensitivity to halo- thane and caffeine compared to normal controls. Ac- cording to the European Malignant Hyperthermia Group's protocol (5), the diagnosis of MHS requires an increase in baseline tension of at least 0.2 g at 2% halothane or less and 2 mmol/l caffeine or less. Even wit 11 this standardised protocol, further specifications seem necessary.

Kwent meetings of the European Malignant Hyperthermia Group have shown the need to mrasure the actual halothane concentration in the tissue bath rather than to depend blindly on the no- minal halothane concentration of the vaporizer ( 6 ) . Also, because caffeine exists as the pure base or the morr soluble citrate or benzoate complexes, it sermed worthwhile to investigate whether these three preparations produce the same amount of contrac- ture when used in equal caffeine concentrations. Be- cause the number of muscle strips necessary for this experiment is difficult to obtain during muscle bi- opsy in humans, a rat model was used.

MATERIAL AND METHODS Three groups of five rats were studied. Male Wistar rats weighing 250 to 400 g were anaesthetized with nembutal injected into the peritoneal cavity. The left upper quadrant of the diaphragm was dissected and four strips (2-3 mm wide and 15-25 mm long) werr taken from each rat.

The muscle strips were then mounted in the tissue bath, which was continuously perfused with Krebs-Ringer solution at 37°C and carboxygenated (5) . One end of the muscle strip was clamped rigidly and the other attached to a strain gauge connected to an amplifier/ recorder system. The muscle strip was stretched to 2 g and left for 15 min to stabilise. The muscle specimens were electrically stimulated by 1 ms supramaximal stimuli at a frequency of 0.2 Hz to prove viability. After obtaining a stable baseline, the bath was perfused with increasing concentrations - I , 2, 4 and 6 mmol . I - ' of caffeine citrate, caffeine benzoate or caffeine pure base. Each concentration was maintained for at least 3 min or until a plateau of maximal tension was obtained.

The caffeine preparations used were: caffeine, free base, biochem- ical grade (97.4% pure when assayed spectrophotometrically at 274 nm in ethanol) (from BDH Chemicals Ltd., Poole, Dorset, England); caffeine citrate, B.P. grade (49.3 to 50.4O6 caffeine) (from Allied Pharmaceutical Distributors, Dublin, Ireland), and caffeine benzo- ate, sodium salt, (from Sigma Chemical Co., St. Louis, MO, U.S.A.).

Both pH and ionized Ca'' were measured at the different conren- trations of the three caffeine preparations in the Krebs solution. pH was measured directly in the tissue bath using a Radiometer PHM82 meter with a combination electrode. Ionized Ca2' was measured ar 37°C using a Radiometer F2112 calcium electrode and PHM62 researrh meter. The electrode was calibrated with standard calcium solutions in the range 0.1 to 10 mmol.l- ' .

Statistical analysis of the results was carried out using a paired Student &test; differences were judged significant when P was < 0.05.

Page 2: The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base?

542 L. HEYTE

The experiments were conducted with the approval of the Ethical Committee of hntwerp University Hospital and by licence fi.om the Ministry of Health, Dublin.

RESULTS Fig. 1 shows the typical cumulative in vitro contracture response of rat diaphragm to caffeine pure base (mean s.d.). Only a very small response of 0.04 g f0 .04 was found at 1 mmol.1-l caffeine but the generated tension steadily increased thereafter as the caffeine concentrations were increased to 6 mmol 1 - I . 'The three different preparations of caffeine were not compared at higher concentrations as only the lower concentrations, 1, 2, 4 and possibly 6 mmo1.1-', are used in the diagnosis of M H susceptibility in humans.

The tension generated at 1 and 2 mmol. 1 - ' caffeine was similar for caffeine benzoate and the free base (Fig. 1) . At higher concentrations a statistically significant difference was found, as shown in Fig. 1. At 4 mmol . 1 - caffeine benzoate generated only 66% and at 6 mmol . 1 - ' only 3204 of the tension generated by caffeine pure base. Caffeine citrate did not produce a contracture at 1 or 2 mmol.1-' and generated less tension at the higher concentrations when compared to the pure base, as is shown in Fig. 2. The differences in tension were statistically significant at all caffeine concentrations tested.

Caffeine free base and caffeine benzoate did not alter the pH of the carboxygenated Ringer solution. On the other hand, a decrease in pH was found with increasing caffeine citrate concentrations (Fig. 3 ) .

Caffeine citrate was also found to have a pro- nounced effect on the ionized calcium concentration

:1/ ol 0 4

i 0 v) z w +

0 1 2 3 4 5 6

CAFFEINE, rnM Fig. I . Comparison of rrsponse ol'rat diaphragm to cumulative doses of caffeine pure base [El 1 dnd to caffeine benzoate [*I. Values are mean s.d. for 5 independent experiments. Bars denote I x s.d. and asterisk indicates significant dillererice at P < 0.05.

:NS ET AL.

of the Krebs solution in the tissue bath, even after pH was readjusted to 7.4, as shown in Fig. 4. The free calcium ion concentration dropped to 50% of the con- trol value at 2 mmol.1-' caffeine citrate. Neither caf- feine free base nor caffeine benzoate altered the free calcium ion concentration of the Krebs solution.

DISCUSSION CaKeine free base or anhydrous caffeine (MW 194.2) is poorly soluble in water, only 1 in 60. For practical purposes it is often rendered more soluble by the ad- dition of an equal quantity of citric acid or sodium

" 1

ol

i s v) z w k

O 6 I

/ * /

0 1 2 3 4 5 6

CAFFEINE, rnM Fig. 2. Comparison of rrsponse of rat diaphragm to cumulative doses of caffeine pure base [ 1 and caffeine citrate [ *]. Values are mean r s.d. for 5 independent experiments. Bars denote I x s.d. and asterisk indicates significant difference at P< 0.05,

0 2 4 6 n 10 CAFFEINE CITRATE, mfl

Fig. 3. Effect of caffeine citrate on the pH of the oxygenated (95", oxygen, 5% carbon dioxide) Ringer at 37°C. Mean values for 2 experimrnts.

6 . 2 l . ' " . ' . ' '

Page 3: The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base?

CAFFEINE CONTRACTURE TEST 543

benzoate. If the differences in molecular weight are taken into account, a stock solution and the subsequent dilutions which contain exactly the same concen- trations of caffeine as the free base solution can rapidly and easily be prepared.

Theoretically, as the same concentration of caffeine is used, one would expect the same cumulative contrac- ture response with increasing caffeine concentrations in the three groups. However, we found the contractures produced by caffeine benzoate at 4 and 6 mmol.1-l and especially by caffeine citrate at all concentrations tested to be smaller than those produced by caffeine free base at equimolar concentrations. The citric acid and sodium benzoate seem to possess physical or chemical properties that attenuate the response of the rat diaphragm muscle to cumulative doses of caffeine.

Canine citrate modifies both pH and the free cal- cium ion concentration. The pH drops with increasing conceri trations of caffeine citrate and reaches approxi- mately 7.00 at 4 mmol 1-’ caffeine citrate. Nakamu- ra & Schwartz (7) have demonstrated that acidosis reduces both the release and uptake of calcium by striated mucle sarcoplasmic reticulum. Fabiato & Fab- iato (8) have shown in skeletal muscle that the maximum tension decreased 30% when the pH was lowerd from 7.40 to 6.20. Furthermore, they found that decreasing the pH from 7.40 to 6.20 increased the free calcium concentration required for the myofila- ments to develop 50% of the maximum tension by a factor nf 5 in skinned cardiac cells and by a factor of 3 in skeletal muscle cells.

Simultaneously with the fall in pH, the free calcium

on concentration drops sharply and is reduced to 50% of the original concentration when 2 mmol 1-’ caffeine citrate is added to the test bath (Fig. 4). Contracture responses to both halothane and caffeine have been found to be significantly decreased when the calcium concentration in the bath is lowered (9). Likewise Cota & Stefani (10) have demonstrated that external calcium has a supporting role in maintaining tension during potassium-induced contractures. Additionally, reduction of external free calcium ion concentration increases the mechanical threshold for pulses shorter than 20 ms (1 1) . All these elements suggest that exter- nal free calcium ion may play a role in the tension generation.

Caffeine free base and the sodium salt of caffeine benzoate produce neutral solutions when dissolved in aqueous media, and thus do not influence the pH in the test bath. Similarly, the free calcium ion concen- tration is not altered by adding caffeine benzoate or the free base. Nevertheless, the benzoate moiety does show an influence on caffeine-elicited contractures: the higher the concentration of caffeine benzoate, the more the tension generation seems to be inhibited as com- pared to the tension generated by caffeine free base. Bianchi showed in 1968 (12) that benzocaine (ethyl p- aminobenzoate) blocks the caffeine-induced contrac- ture; 1.2 mmol.l-’ markedly depressed the 5 mmol 1-’ caffeine contracture and 2.4 mmol - 1-’ com- pletely blocked it. Friedman et al. (13) in 1974 investi- gated the structural aspects of the effect of the ethyl aminobenzoates on caffeine contracture. The ethyl p- aminobenzoate and ethyl m-aminobenzoate (Fig. 5)

* OXY GENATEO ONLY 0-C-O- C2Hs 0 - C - 0 - C2Hg I I I OXYGENATED A N 0 pH AOJUSTEO

0.2

0.0

- I I I

0 2 4 6

H Ethyl benzoate

0 - C - 0 - C2Hs I

N H2 Ethyl-p amino benzoate

o=c-0-Na

\

CAFFEINE C ITRATE, mt’l H . .

Ethylo-amino benzoate Sodium benzoate Fig. 4. k:lTect of caffeine citrate on the ionised calcium concentration of RinKrr solution which was oxygenated 101 and on a separate - snmplt which was oxygenated after readjustment of the pH to 7.4 l o ] .

Fig. 5. Structural formulae of the ethylaminobenzoates (including benzocaine) compared to sodium benzoate.

Page 4: The caffeine contracture test for malignant hyperthermia: caffeine citrate, caffeine benzoate or caffeine free base?

544 L. HEYTENS E’I’ AL.

markedly depressed contracture. However, when the amino group was placed in the position ortho to the carbonyl group - thus resulting in steric hindrance of the latter group - an increased caffeine contracture was found. These observations indicate that the carbonyl group of benzocaine is the blocking locus for the caf- feine contracture. The structure of sodium benzoate is very similar to the structure of benzocaine. For this reason it is probable that the same mechanism ac- counts for the reduced caffeine contracture when so- dium benzoate is added to the caffeine.

In conclusion, caffeine benzoate and caffeine citrate generate less tension in rat diaphragm than caffeine pure base. Caffeine citrate decreases the pH and the free calcium ion concentration, and together these are believed to be responsible for the decrease in contrac- ture development. The depression of the caffeine con- tracture by sodium benzoate seems to be due to a direct antagonizing effect of the carbonyl group on the caffeine action on muscle, i.e. on release of Ca2 + . The results indicate that only the caffeine free base should be used in the in vitro contracture test for MH.

REFERENCES Brownell A, Paasuke R, Elash A et al. Malignant hyperthermia in Duchenne muscular dystrophy. Anesthesiology 1983: 58: 180-181. Nelson T, Flewellen E, Cloyna D. Spectrum of susceptibility to malignant hyperthermia - diagnostic dilemma. Anesth Analg

Ellis F R , Halsall P J, Harriman D G F. The work of the 1983: 62: 545-555.

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Leeds Malignant Hyperpyrexia Investigation Unit, 1971-1984. Anaesthesia 1986: 41: 809-815. Ording H, Ranklev E, Fletcher R. lnvrstigation of malignant hyperthermia in Denmark and Sweden. Br J Anaesfh 1984: 56:

European Malignant Hyperthermia Croup. A protocol for thr investigation of malignant hyperthermia ( M H ) susceptibility. Hr J Anaesth 1984: 56: 1267-1269. Brding H. Diagnosis of susceptibility to malignant hypcrthermia in man. Er J Anaesth 1988: 60: 287-302. Nakamura Y, Schwartz A. The influence of hydrogrn ion con- centration on calcium binding and rrlease by skeletal musclc sarcoplasmic reticulum. J Gen Phvsiol 1972: 59: 22-32. Fabiato A, Fabiato F. Effects of pH on the myofilaments and thr sarcoplasmic reticulum of skinned cells from cardiac and skelrtnl muscles. J Physiol 1978: 276: 233-255. Nelson T E, Chausmer A B. Calcium content and rontracturc in isolated muscle of malignant hyperthermic pigs. J Pharnmd Exp Ther 1981: 219: 107-11 I . Cota C , Stefani E. Effects of external calcium reduction on the kinetics of potassium contractures in frog twitch muscle fihrcs. J Physiol 1981: 317: 303-316. Chiarandini D J, Sanchez J A, Stefani E. Efiect of calcium withdrawal on mechanical threshold in skeletal muscle fihrrs 0 1 the frog. J Physiol 1980: 303: 153-163. Bianchi C P. Pharmacological actions on excitation-coritractioii coupling in striated muscle. Fed Proc 1968: 27: 126-131. Friedman H A, Bianchi C P, Weiss S J. Structural aspects of thc effect of ethylaminobenzoates on caffeine contracture. .7 Pharmar- 01 Exp Thcr 1974: 189: 423433.

1183-1 190.

Address: Professor J . J . A . Heffron Department of Biochemistry University College National University of Ireland Cork Ireland