Sakakihara et al.: Microassay for dichloroacetate by gel-penneation chromatography 79

Eur. J. Clin. Chem. Glin. Biochem.Vol. 32, 1994, pp. 79-83© 1994 Walter de Gruyter & Co.

Berlin · New York

A Rapid Microassay for Dichloroacetate in Serumby Gel-Permeation Chromatography

By Y. Sakakihara, G. Nakamura, Y. Tokoeda, T. Abe and S. Kamoshita

Department of Pediatrics, Faculty ofMedicine, The University ofTokyo

(Received April 5/November 8, 1993)

Summary: We have developed a novel, rapid microassay for dichloroacetate in the serum. The serum sample isdirectly injected into a gel-permeation high-performance liquid chromatography apparatus. The peak of dichlo-roacetate appears after a giant protein peak. The method requires a very small amount of serum (10 ), and theanalysis time is short (20 min). Using this micro method, we measured the serum concentrations of dichloroacetatein healthy adult volunteers and paediatric patients with congenital lactic acidosis. Although the effect of dichlo-roacetate on the neurological manifestations of congenital lactic acidosis has not been proved to be beneficial, thepotential usefulness of dichloroacetate in refractory lactic acidosis in cardiac and respiratory failure has beenrecognized, and human äs well äs animal studies have been undertaken in many laboratories. To prevent possibleside effects of dichloroacetate, it has been recommended that the minimal effective dose be used. Our microassaymethod is useful for both human and animal experiments, even after administration of minimal doses.

Introduction

Dichloroacetate äs the sodium salt has been used for thetreatment of hyperglycaemia, lactic acidosis, and hyper-cholesterolaemia (1—7). Although dichloroacetate hasmany metabolic effects on glucose, lipid and amino acidmetabolism, its main pharmacologic effect has beenshown to be the activation of the pyruvate dehydrogen-ase1) complex by inhibiting pyruvate dehydrogenase ki-

The clinical usefulness of dichloroacetate has been ham-pered by its possible toxic and mutagenic effects (7).Peripheral neuropathy and testieular atrophy have beenreported in humans äs well äs animals after chronic ad-ministration of dichloroacetate (12). Recently, however,dichloroacetate has been reevaluated in response to themany reports on its efficacy in the refractory acidosisencountered in emergency medicine, such äs cardiac ar-rest and septic shock (13 — 15).

Although there have been few reports on the acute tox-icity of dichloroacetate, it has been recommended thatthe minimal effective dose be used in order to preventpossible adverse effects (7). Dichloroacetate has beenmeasured by gas-chromatography (11), but the measure-ment is time-consuming and requires 2 ml of blood. Be-cause of these technical difficulties, blood concen-trations of dichloroacetate have not been routinely meas-ured.

In the present study, we have developed a rapid micro-method for dichloroacetate measurement using gel per-meation chromatography. With this micromethod, wehave investigated the pharmacokinetics of dichloroacet-ate by measuring its level in serum in various patientswith lactic acidosis, äs well äs in healthy adult volun-teers. Since the new method requires a very small am-ount of serum (10 ), and needs no extraction procedurefor sample preparation, it is suitable for measuringserum concentrations of dichloroacetate in human äswell äs animal experiments.

!) Enzymes:Pyruvate dehydrogenase, EC 1.2.4.1; Pyruvate dehydrogenase kin-ase, EC 2.7.1.99.

Materials and MethodsDichloroacetate (sodium salt) was purchased from Tokyo Kasei(Tokyo). The other cheraicals were of the highest quality availableand obtained frorn Standard sources.

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 32, 1994 / No. 2

80 Sakakihara et al.: Microassay for dichloroacetate by gel-permeation chromatography

Blood samplcs were drawn from patients with lactic acidosis ofvarious actiologies and from normal adult volunteers. The basicdisorders of thc patients and doses of dichloroacetate are summar-ized in table 1. Informed consent was obtained from the parents ofthc patients betöre dichloroacetate administration. The volunteerswerc given 35 mg/kg of dichloroacetate orally. Blood samples weredrawrfbeforc and after oral dichloroacetate loading at 30 minutesintervals. Serum was separated immediately and kept frozen at-20 °C until analysis.

A small amount (15 to 25 ) of serum was directly injected intoa high-performance liquid chromatography (HPLC) apparatus. Agel-permeation HPLC column (Asahipak GS-320, Asahi Kasei, To-kyo) was equipped with a pump (Waters, Model 6000A) and UVspectrophotometer (Soma UV detector, Soma Kogaku, Tokyo). Thecolumn was eluted with 0.01 mol/1 ammonium acetate (pH 4.0)/acetonitrile (9+1, by vol.) at a flow rate of 2.0 ml/min, and theeluate was monitored at 220 nm. The quantitation of dichloroacet-ate was carried out by comparing the peak height of dichloroacetatewith the calibration curve constructed by plotting the peak heightsof various known amounts of dichloroacetate.

Dichloroacetate was also measured by gas-chromatography accord-ing to the method of Wells et al. (11) with trichloroacetic acid ästhe internal Standard.

Results

The new Chromatographie method ofdichloroacetate measurement

The chromatogram of normal serum is shown in figurel a. In the gel-permeation column, molecules whosesizes are greater than Mr 40 000 are excluded from thecolumn bed, and the smaller molecules are retained inthe column for various times according to the interactionbetween the molecules and the packing material. Thusthe chromatogram of normal serum consists of a giantpeak which represents the serum components with mol-ecular mass greater than 40000 (mostly proteins) andseveral unknown peaks following it. The chromatogramof the serum from a volunteer after dichloroacetate in-take is shown in figure Ic. As shown in the figures, adistinct peak appeared at 12.2 min. A peak with exactlythe same retention time was observed when serum pre-

Tab.l

Pa-tientNo.

1

2

3

4

Profiles

Age(a)

8 5/12

3 8/12

8 6/12

9/12

of the patients.

Sex Diagnosis

$ Pyruvate dehydrogenasecomplex deficiency

S Pyruvate dehydrogenasecomplex deficiency

? Mitochondrial encephalo-pathy, lactic acidosis andstroke-like episode(MELAS)

? Leigh encephalopathy

Dose ofdichloro-acetate(mg/kg · d)

80

40

30

30

10 20 0 10Retention time [min]

20

V

10 20 0 10

Retention time [min]

20

Fig. l Chromatograms of dichloroacetate in serum.(a) Chromatogram of normal serum. Note the giant peak irnmedirately after the solvent peak.(b) Chromatogram of normal serum mixed with authentic dichlo-roacetate.(c) Chromatogram of the serum of a volunteer after a single oraldichloroacetate load.(d) Chromatogram of the serum from Patient 1.

viously supplemented with authentic dichloroacetatewas analysed (fig. Ib). The chromatogram of serumfrom Patient l is shown in figure Id. The calibrationcurve constructed by injecting various known amountsof authentic dichloroacetate showed a linear relation'be-tween 0 to 500 mg/1 äs shown in figure 2. The minimalineasurable concentration was 5 mg/1 when 25 ofserum was injected, but a much lower coricenträtion wasmeasurable by increasing the amount injected, orby in-creasing the sensitivity of the UV detector. To assessthe reproducibility of the method, we injected replicatealiquots of the sample. The coefficient of Variation (CV)was found to be very small (CV = 2.4%, n = 8).

We measured the levels of dichloroacetate in 9 serumsamples by conventional gas chromatography concomi-

i:

l

Eur. J. Clin, Chem. Clin. Brachem. / Vol. 32,1994 /No. 2

Sakakihara et al.: Microassay for dichloroacetate by gel-permeation chromatography 81

10

8-o

Ο 50 100 150 200 250Dichloroacetate [mg/1]

Fig. 2 Calibration curve of dichloroacetate. Serum samples withknown concentrations of dichloroacetate (10, 50, 100, 200 mg/1)were prepared by adding authentic dichloroacetate to serum froma healthy adull volunteer. A 25 μΐ aliquot of each sample was in-jected into HPLC. Good linearity was obtained between 10 to 200mg/1 (y = 0.04x + 0.045, r = 0.997).

tantly with the new micromethod (l 1). As shown in fig-ure 3, there was a good correlation between the resultsobtained from the two methods.

Finally, the specificity of the method was ascertainedby injecting the sera from 10 patients who were takingdifferent drugs (antiepileptic drugs, antibiotics) into theHPLC. No peaks were observed around the retentiontime of the dichloroacetate peak (data not shown).

Pharmacokinetics of dichloroacetate inhealthy adult volunteers

Using the micromethod, we investigated the pharmaco-kinetics of dichloroacetate in healthy adults. The timecourse of the levels of dichloroacetate in two volunteersis shown in figure 4. The dichloroacetate was absorbedquickly and reached a peak concentration within 30—60minutes. The level then dropped rapidly, and by 5 hoursdichloroacetate had disappared from the serum. The halflife of dichloroacetate eliminatipn from the serum wascalculated to be around 30 min by the rnodified methodof Wagner & Nelson (16).

S %QO

250

200

150

100

50

50 100 150 200 250Dichloroacetate [mg/1]Gel-permeation chromatography

Fig. 3 Correlation between the new micromethod and gas chrom-atography. A good correlation was obtained between the two me-thods (y = 1.07x - 0.74, r « 0.992).

100 200 300Time [min]

400

Fig. 4 Concentrations of dichloroacetate in the sera from healthyadult volunteers after a single oral dose of dichloroacetate (35 mg/kg). The half lives of elimination are 0.534 h (-D-) and 0.573 h(-0-).

Measurement of serum dichloroacetate levelsin patients

The typical time course of serum dichloroacetate con-centrations is shown in figures 5a and 5b. In patients l,2 and 3, dichloroacetate had been orally administratedfor various periods previously. In Patients 2, 3 and 4, theattained peak levels of dichloroacetate were different,

100 200 300

Time [min]

400

125

^100-I

50-

25-S

100 200 300 400 500 600Time [min]

Fig. 5 Concentrations of dichloroacetate in the sera from thepatients.(a) Patient l (-·-)(b) Patients 2 (-O-), 3 (-·-), and 4 (-O-).In Patients l, 2 and 3, dichloroacetate had already been adminis-tered for various times before the measurements. Thus the levels ofdichloroacetate before oral administration (at time 0) were not zero.The half-lives of dichloroacetate for Patients l, 2, and 3 were245 h, 0.18 h and 0.87 h respectively.

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 32,1994 / No. 2

82 Sakakihara et al.: Microassay for dichloroacetate by gel-permeation chromatograpby

although the doses of dichloroacetate per body weightwere similar. It was also shown that the ratio of dose toblood level was not linear, äs indicated by the very highlevels of dichloroacetate in Patient 1. The very pro-longed half-life of dichloroacetate in Patient l suggestsan impaired elimination of dichloroacetate from the cir-culation in this case.

Discussion

We demonstrated that the micromethod for dichloroacet-ate measurement is useful for monitoring serum dichlo-roacetate. Once the HPLC apparatus was set up, it usu-ally took only 20 minutes to obtain the results. In theconventional gas-chromatographic method, it wouldtake at least several hours, since deproteinization andderivatization of dichloroacetate must be performed be-fore the Chromatographie measurement (11). Addition ofinternal Standard is also required in the gas^chromato-graphic method. Furthermore, the new method requiresmuch less sample: the gas-chromatographic method re-quires l ml of serum, whereas äs little äs 10 is enoughfor the micro method. The serum contained in a glasscapillary is sufficient for the measurement of dichlo-roacetate by the new method. We are therefore able tomonitor dichloroacetate concentrations even in smallanimals.

The pharmacokinetics of dichloroacetate determined bythe micro method are comparable with the previouslyreported values obtained with a single intravenous injec-tion (5, 11, 17, 18). Although the accurate calculation ofthe half-life was difficult when the drug was admini-strated orally, we estimated the approximate half life of

dichloroacetate by a modified calculation method ofWagner & Nelson (16). In healthy adults, the half-lifewas approximately 30 minutes, although the calculatedhalf-life of dichloroacetate was much longer than this inPatient 1. It has been reported that the elimination ofdichloroacetate from the circulation follows Saturationkinetics (11), and tissue accumulation of dichloroacetateoccurs when dichloroacetate is administered for a longtime. Adverse effects such äs polyneuropathy and tes-ticular atrophy have been reported after chronic dichlo-roacetate treatment in human and animal studies (7, 12).Cönsidering the unpredictability of exact blood levels ofdiehloroacetate, it is pertinent to measure them inpätients treated with dichloroacetate. The fact thatdichloroacetate therapy has not yet been accepted äs aStandard therapy for lactic acidosis is mainly due to itspossible toxic side-effects. It is important to maintainthe reported minimum therapeutic level (130 mg/1) ofdichloroacetate (11). Although it is undoubtedly effec-tive in lowering blood lactate and pyruvate in pätientswith congenital lactic acidosis, it has been reported toshow little effect in improving existing neurologicSymptoms (7). Recently, however, its potential clinicalusefulness has been reappraised, especially in criticalcare medicine (13 — 15). It has been demonstrated thatdichloroacetate is effective in correcting severe acidosisrefractoiy to the conventional sodium bicarbonate ad-ministration. It has also been shown that it has a protec-tive effect against myocardial ischaemic damage. Toconfirm its potential clinical usefulness, many labora-tories have been eonducting animal experiments to de-termine its efficacy and safety. Our micromethod is suit-able for monitoring blood dichloroacetate concentrationsin animal experiments äs well äs in clinical trials.

References

1. Stacpoole, P. W., Moore, G. W. & Komhauser, D. M. (1978)Metabolie efFects of dichloroacetate in pätients with diabetesand hyperlipoproteinemia. N. Engl. J. Med. 298, 526-530.

2. Coude, F. X., Saudubray, J. M, DeMaugre, F., Marsac, C.,Leroux, J. P. & Charpentier, C. (1978) Dichloroacetate äs treat-ment for congenital lactic acidosis. N. Engl. J. Med. 299,1365-1366.

3. McKhann, G., Francois, B. & Evrard, P. (1980) Long term useof low doses of dichloroacetate in a child with congenital lacticacidosis. Pediatr. Res. 14, 167.

4. Park, H. & Areff, A. 1. (1982) Treatment of lactic acidosiswith dichloroacetate in dogs. J. Clin. Invest. 70, 853-862.

5. Stacpoole, P. W., Harman, E. M, Curry, S. H., Baumgartner,T. G. & Misbin, R. L (1983) Treatment of lactic acidosis withdichloroacetate. N. Engl. J. Med. 309, 390-396.

6. Aynsley-Green, A, Weindling, A. M., Soltesz, G. & Jenkins,P. A. (1983) Transient lactic acidosis and hyperaläninaemiaassociated with neonatal hyperinsulinemic hypoglycaemia:The effect of dichloroacetate (DCA). Eur. J. Pediatr 141114-117.

7. Stacpoole, P. W. (1989) The pharmacology of dichloroacetate,Metabqlism 38, 1124-1144.

8. Whitehouse, E. & Rändle, P. J. (1973) Activation of pyruvatedehydrogenase in perfused rat heart by dichloroacetate. Bio-chem. J. 134, 651 -653.

9. Abernayor, E., Kovachich, G. B. & Haugaad, N. (1984) Effectof dichloroacetate on brain pyruvate dehydrogenase. J. Neurp-chem. 42, 38-42.

10. Kuroda, Y., Toshima, K., Watanabe, T., Kobashi, H., Ito, M.,Takeda, E. & Miyao, M. (1984) Effects of dichloroacetate onpyruvate metabolism in rat brain in vivo. Pediatr. Res. 18,936-938.

11. Wells, P. G., Moore, G. W., Rabin, D., WilJdnson, J. A. &Stacpoole, P. W. (1980) Metabolie effects and pharmacokinet-ics of intravenously administrated dichloroacetate in humans.Diabetologia 19, 109 -113.

12. Stacpoole, P. W., Moore, G. W. & Konihauser, D. M. (1979)Tqxicology of chronic dichloroacetate. N. Engl. J. Med. 300,372.

13. Stacpoole, P. W., Lorenz, A. C., Thomas, R. G. & Harman, E.M. (1988) Dichloroacetate in the treatment of lactic acidosis.Ann. Int. Med. 108, 58-63:,

Eur. J. Clin. Cherri. €Hn. Biochem. / Vol. 32,1994 / No. 2

Sakakihara et al.: Microassay for dichloroacetate by gel-permeation chromatography 83

14. Graf, H., Leach, W. & Arieff, A. J. (1985) Effects of dichlo- 18. Curry, S. H., Chu, P. I., Baumgartner, T. G. & Stacpoole, P.roacetate in the treatment of Hypoxie acidosis in dogs. J. Clin. W. (1985) Plasma concentration and metabolic effects of intra-Invest. 76,919-923. venous sodium dichloroacetate. Clin. Pharmacol. Ther. 37,

15. Burns, A. H., Gioimo, M. E. & Summer, W. R. (1986) Dichlo- 89-93.roacetic acid improves in vitro myocardial function followingin vivo endotoxin administration. J. Crit. Care 7, 11-17. vnW Q v L··»,

16. Wagner, J. G. & Nelson, E. (1964) Kinetic analysis of blood Yoicni ^aKaKiüaralevels and urinary excretion in the absorptive phase after single Department of Pediatricsdoses of drug. J. Pharmaceut. Sei. 53, 1392-1403. Faculty of Medicme

17. Lukas, G., Vyas, K. H., Brindle, S. D., LeSher, A. R. & The University of TokyoWagner, W. E. (1980) Biological disposition of dichloroaeetate 7-3-1 Hongo, Bunkyo-kuin animals and humans after intravenous administration. J. Tokyo 113Pharmaceut. Sei. 69, 419-421. Japan

Eur. J. Clin, Chem. Clin, Biochem. / Vol. 32,1994 / No. 2