Determination of 5-aminolevulinic acid in biological samples by high-performance liquid chromatography

ANALYTICAL

BIOCHEMISTRY

149, 29-34 (1985)

Determination of 5Aminolevulinic Acid in Biological Samples by High-Performance Liquid Chromatography HANS-ULRICH Fachrichtung

Biochemie

MEISCH,’

WOLFGANG REINLE, AND URSULA WOLF

der Universitiit

des Saarlandes,

D-6600

Saarbriicken.

West Germany

Received December 10, 1984 5-Aminolevulinic acid (ALA), the common precursor of all naturally occurring tetrapyrroles, forms a stable condensation product with 2-amino-3-hydroxynaphthafene which can be identified by its fluorescence. Separation of the compound by reversed-phase high-performance liquid chromatography on RPC-18 columns allows its detection down to the picomolar range and can be successfully applied for ALA analysis in small biological samples. The reaction product of ALA with 2-amino-3-hydroxynaphthalene has been synthesized and characterized. 0 1985 Academic

Press, Inc.

KEY WORDS: 5-aminolevulinic cence: HPLC; Chlorella fusca.

acid: 2-amino-3-hydroxynaphthatene;

5Aminolevulinic acid (ALA)2 is the first committed intermediate during the biosynthesis of all naturally occurring tetrapyrroles like hemes, chlorophylls, corrins, or bile pigments (1). The quantitative determination of ALA is of considerable clinical interest since ALA biosynthesis is an important regulatory step during tetrapyrrole formation, sometimes giving rise to abnormal ALA levels in biological fluids during pathological conditions, like porphyrias or lead poisoning (2). On the other hand, ALA synthesis is also the ratelimiting step for chlorophyll biosynthesis in green plants and photosynthetic bacteria (3), its formation being critically controlled by light (4). Quantitative ALA determination is therefore often used as a tool for investigations on ALA and chlorophyll biosynthesis and their regulation (for review see (1)). For this purpose, the method of Mauzerall and Granick (5) is commonly used: ALA is allowed to react with a 1,3-diketone (ethyl acetoacetate ’ To whom correspondence should be addressed. ’ ALA, 5-aminolevulinic acid: AHN. 2-amino-3-hydroxynaphthalene; AHN-ALA, 5-amino-4-(2)-imino-3hydroxynaphthyl-levulinic acid; TCA, trichloroacetic acid; DMSO. dimethyl sulfoxide. 29

derivatization: fluores-

or acetylacetone), followed by a condensation of the resulting pyrrole with 4-(N)-dimethylaminobenzaldehyde (Ehrlich’s reagent). The red color is then used for quantitation. Since Ehrlich’s reagent is not very specific and undergoes a number of color reactions with other compounds like pyrroles and a-aminoacetone (6), the method fails to bring about reliable results in biological samples, where small amounts (~4 nmol) of ALA have to be determined. In studies on chlorophyll and ALA biosynthesis in connection with the trace metals vanadium and iron (7,8), we investigate the enzymatic conversion of 2-oxoglutarate or Lglutamate into ALA. So we need a very sensitive method for the detection of small amounts of ALA which may have been formed during the assay. The method of Mauzerall and Granick (5) proved to be insensitive for this purpose. We therefore describe a new method which allows the determination of nano- to picomolar amounts of ALA in biological samples after condensation with 2-amino-3-hydroxynaphthalene and separation of the fluorescent derivative by HPLC. 0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproducbon in any form reserved.

30

MEISCH,

EXPERIMENTAL

PROCEDURES

REINLE,

AND

WOLF

fluorescent compound being followed by uv irradiation. The fluorescent fractions were pooled, evaporated, and separated again on Chromatography. The chromatographic silica gel as before. Evaporation of the pure analyses were performed with a high-performance liquid chromatograph equipped with fraction yielded the yellow ALA derivative. a Waters 6000 A pump (Waters Assoc., MilALA analysis. Aqueous samples or algal ford, Mass.), a Rheodyne injection valve, a extracts containing up to 1 pmol ALA in 10 Perkin-Elmer 65O/LC fluorescence detector ml were treated with 10 pmol AHN (1.6 mg set at 370 nm (excitation) and 558 nm (emis- AHN in 2 ml of ethanol) and brought to pH sion), and a multirange recorder. A reversed- 10 with 1 M NaOH. The condensation was carried out at 60°C (90 min). The excess phase system was used consisting of a Merck HIBAR RPC-18 column (25 cm X 4 mm, AHN was then extracted with 3 X 3 ml of Lichrosorb C-l 8, particle size 5 pm), which diethyl ether. The aqueous solution was acidified to pH 4 with 1 M HCl and then extracted was eluted at 25°C with a degassed mobile phase of methanol/water/acetic acid, 701301 as before. The latter extracts were combined 0.5). Samples of 20 ~1 were injected, and the and evaporated to dryness and the residue was redissolved in a defined volume of methflow rate was 1.5 ml/min. anol (200 ~1 or more, dependent on ALA Spectroscopic measurements. Ultraviolet concentration), this solution now being ready spectra were obtained with a Cat-y Model 17D spectrophotometer, fluorescence emis- for HPLC analysis. Preparation of biological samples. Extracts sion and excitation spectra were performed with a Perkin-Elmer MPF-44 A fluorescence were prepared from the unicellular green alga spectrophotometer. ‘H NMR spectra were Chlorella fusca. strain 2 l l-8b (Collection of recorded in a 90-MHz Bruker HX-90 instru- Algae, Gottingen), the organisms having been ment (Bruker, Karlsruhe, West Germany), autotrophically cultivated as described earlier (7). After 3 days of growth, ammonium and mass spectra were obtained in a Varian MAT 3 11 spectrometer at 70 eV and 190°C. levulinate (34 mM, pH 6.5) was added to the Chemicals and synthesis of the ALA deriv- nutrient medium, and the algae were allowed ative. AHN was purchased from EGA to grow for a further 24 h. During that time, Chemie, Steinheim; all other chemicals were cells from 50-ml samples were harvested by from Merck, Darmstadt. The derivative of centrifugation (lSOOg, 5 min), treated with 10% trichloroacetic acid (TCA), and centriALA with AHN was synthesized as follows: 50 mg of ALA - HCl in 80 ml of water and fuged again. Any intracellular ALA was now 50 mg AHN in 20 ml of ethanol were found in the supernatant. 2 ml samples of combined, brought to pH 10.0 with 1 M these extracts served as the matrix for further NaOH, and kept for 90 min at 60°C. Then ALA determination. the solution was extracted with 5 X 10 ml of RESULTS diethyl ether in order to remove any excess of the reagent. The aqueous phase was Synthesis of ALA Derivative and brought to pH 4.0 with 1 M HCl and extracted Structural Identification again with diethyl ether as above. The latter extracts were combined and evaporated to The excitation maximum for the ALA dryness. The residue was dissolved in a minderivative (AHN-ALA) was at 372 nm and imum amount of isopropanol and applied to the emission maximum was at 558 nm a silica gel column (Kieselgel 60, Merck; (Fig. 1). column 1.5 X 30 cm). Elution was performed The alkaline condensation of ALA with with ispropanol, fractionation of the green AHN required the determination of an op-

CHROMATOGRAPHIC

SAMINOLEVULINIC

ACID DETERMINATION

31

cence detection). The compound was further characterized by mass spectroscopy (Table 1). Additionally, a ‘H NMR spectrum was performed in DMSO: ‘H NMR (DMSO) 6 (ppm) = 7.7-7.5 (m, 6H, aromatic H), 5.4 (s, 1/2H, OH/OH& 3.5 (s, broad, 5/6H, NH/NH*, 2CH2), 1.9 (s, 2H, CHz). According to these spectral data, the structure of AHN-ALA (III) is proposed as shown in Scheme 1. With respect to the ‘H NMR, the broad FIG. 1. Fluorescence emission spectra of AHN (---) and of the condensation product of AHN with low-field multiplet represents the aromatic ALA (-). protons of the two condensed rings. The signal at 5.4 ppm can be attributed to a hydroxyl proton. This assignment is suptimal pH value. The relative conversion rate ported by the high 6 value which is characby percentage as a function of the pH value teristic of phenolic OH groups, as well as by is indicated in Fig. 2a. It is shown that at pH the fact that an additional spectrum recorded 10 the conversion is nearly complete. in D20 does not show any peak at this Fig. 2b illustrates the influence of varying position as one would expect for easily exthe condensation time: At pH 10 quantitative changeable protons. The proton resonance derivatization requires 1 h. A temperature of of the NH2 group appears at 3.5 ppm, show60°C should not be exceeded during the ing the typical broadening caused by quadcondensation, because AHN had been dis- rupol relaxation. In the same parts per million solved in ethanol prior to reaction (see Ex- range the CHz protons neighboring the COOperimental Procedures). and NH2 groups show up. The resonance of To achieve quantitative extraction of AHNthe third CHz group is found at higher field ALA from the aqueous phase by diethyl (1.9 ppm). ether, an optimal pH value of 4 must be According to the mass spectrum, the peak maintained, owing to the amphoteric char- of highest mass (m/e = 269) was attributed acter of the ALA derivative (Fig. 2~). to the M-3 molecular peak which most tenThe condensation of ALA with AHN was tatively was formed by ring closure between also performed under nitrogen. HPLC anal- the phenolic OH group and the amino group ysis showed that an oxygen-free environment of ALA, thus yielding a more stable and does not alter the reaction of AHN with volatile compound. Subsequent decarboxylALA thereby excluding any oxidation of ation at the C-l position of ALA gives rise the aromatic amine prior to its reaction to a highly intense mass peak at 225, indiwith ALA. cating the high stability of this fragment. For its chemical identification AHN-ALA Further removal of a CzH4 group and fragwas synthesized and purified as described mentation of the aromatic ring seem to occur under Experimental Procedures. This refer- with comparable probability as shown by the ence material had a decomposition tempersimilar intensities of the peaks at m/e = 197 ature of 170°C and a Rf value of 0.5 1 in and 186. The subsequent decomposition into thin-layer chromatography on Kieselgel (mo- aliphatic and aromatic fragments leads to the bile phase: toluene/acetic acid, 2/l, fluores- masses 168 and 102. Thus AHN-ALA has

32

MEISCH,

C”fd

REINLE, AND WOLF bl

a

100.

100

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80-

60-

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40-

0 u.a

60

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FIG. 2. Determination of optimal conditions for a quantitative recovery of ALA by condensation with AHN and subsequent extraction of the condensation product into diethyl ether. (a) Variation of pH during condensation; (b) variation of condensation time; (c) pHdependence of the extraction yield. Conditions: 60°C; 0.3 nmol ALA, 6 nmol AHN in 20 mM NaOH/ethanol (2/l); (a) condensation for 1 h, (b) pH 10, (c) condensation for 1 h at pH 10.

been identified as 5-amino-4-(2)-imino-3-hydroxynaphthyl-levulinic acid (III). Separation of ALA Derivative by HPLC The HPLC separation of AHN-ALA (RPC18 column, mobile phase: methanol/water/ acetic acid 70/30/0.5) gave rise to a peak at a retention time of 2.15 f 0.05 min. Remaining amounts of AHN (excitation maximum at 342 nm, emission maximum at 380 nm) were well separated (tR = 10 min).

The condensation of variable amounts of ALA with AHN was performed either in aqueous solution or in the matrix of algal extracts. Since the Schiff base of ALA and AHN is not very stable in aqueous solution, the extraction of AHN-ALA should be performed within 1 h after the condensation step. In diethyl ether or methanol, however, AHN-ALA can be stored for at least 24 h without any detectable loss of fluorescence intensity. Extraction of AHN-ALA, subse-

CHROMATOGRAPHIC

5-AMINOLEVULINIC

ACID

DETERMINATION

33

It was further checked whether the HPLC peak of AHN-ALA was influenced by any MASS SPECTRUM (70 eV, 190°C) OF AHN-ALA unexpected fluorescent compound which m/e value Relative intensity might have been present in algal extracts. For this purpose, the HPLC was also run 289 53.4 with a mobile phase of acetonitrile/water/ 225 100.0 acetic acid (40/60/0.5). At a flow rate of 1.0 197 83.0 186 ml/min, AHN-ALA eluted after a retention 80. I 168 45.6 time of 3.8 min as a single sharp peak which 102 49.2 had the same intensity as when it was mon44 98.3 itored in the system commonly used. In another series of experiments, the new method of ALA analysis was compared to quent HPLC separation, and fluorescence the classic calorimetric reaction of ALA with detection provided the calibration curves as Ehrlich’s reagent (5). Extracts of C. fusca, shown in Fig. 3., the fluorescence being linear grown for some hours in the presence of between 0.15 and 2.5 nmol ALA. The deteclevulinate as indicated under Experimental tion limit was about 40 pmol ALA. In Procedures, normally contain small amounts aqueous solution, the mean relative standard of ALA which can be measured in TCA deviation as provided by lo-fold analyses at concentrations of 0.37, 0.60, 1.56, and 2.40 extracts of the cells (8). As determined by the Ehrlich reaction, cell extracts of C. fusca nmol ALA, respectively, was 5.6%. To determine the completeness of conver- contained after 4 h of cultivation 8.0 & 0.5 pM ALA and after 8 h 10.4 + 0.6 pM ALA sion of ALA to 5-amino-4-(2)-imino-3-hy(mean value of six measurements). Applicadroxynaphthyl-levulinic acid, the synthesized tion of the HPLC method to the same samreference material was used. A comparison of both calibration curves (Fig. 3) resulted in ples yielded 7.4 + 0.4 and 10.8 ? 0.5 PM an average recovery of 86% of the total ALA, respectively, good correspondence of amount of ALA employed. ALA analysis in the two methods. algal extracts was calibrated by standard additions of ALA. As indicated in Fig. 3, ALA DISCUSSION analysis in algal extracts is linear, too, but it is slightly influenced by the matrix: 90% In analogy to the condensation of 4,5recovery of ALA compared to aqueous so- dioxovaleric acid with 2,3-diaminonaphthallutions was found. The statistics of IO-fold ene to form benzoquinoxalene-2-propionic ALA determinations in algal extracts yielded acid, a reaction well described by Porra and a mean relative standard deviation of 5.3%. Klein (9), we condensed ALA with 2-aminoTABLE

1

coo*

COO’

ALA

z-Amino3 -naphthol

I

SCHEME

I

34

MEISCH, REINLE,

AND WOLF

ALA concentration FIG. 3. Calibration curves for the determination HPLC, and fluorescence detection. 0, determination synthetic ALA/AHN derivative; A, determination standard additions of ALA. Conditions: excitation, Experimental Procedures.

3-hydroxynaphthalene under alkaline conditions. The condensation of ALA with 2,3diaminonaphthalene to form benzoquinoxalene-Zpropionic acid, a reaction which was shown to interfere with dioxovaleric acid analysis (lo), could not be used for ALA analysis because this reaction was found to be not quantitative (11). The a-aminocarbony1 function of ALA probably reacts with 2,3-diaminonaphthalene to dihydrobenzoquinoxalene, which disproportionates similar to the dihydroquinoxalenes, yielding tetrahydrobenzoquinoxalene and benzoquinoxalene (12). The derivatization of ALA with AHN to form 5-amino-4-(2)-imino-3-hydroxynaphthyl-levulinic acid is proposed to be a more selective and sensitive method for ALA analysis than the conventional reaction with pdimethylaminobenzaldehyde. Since the new method showed a detection limit of 40 pmol ALA, a loo-times-higher sensitivity compared to the color reaction with Ehrlich’s reagent (detection limit about 4 nmol ALA (5)) can be calculated. Evaporating the ether extract and dissolving the residue in a smaller

of ALA after condensation with AHN, separation by of authentic ALA, X, reference curve obtained from of ALA in TCA extracts of Chlorella fisca after 372 nm; emission 558 nm. For further conditions see

amount of methanol crease in sensitivity.

leads to a further in-

REFERENCES 1. Granick, S., and Beale, S. I. (1978) in Advances in Enzymology & Related Areas of Molecular Biology (Meister, A., ed.), Vol. 46, pp. 33-180, WileyInterscience, New York. 2. Karlson, P. P., Gerck, W., and Gross, W. (1982) Pathobiochemie, pp. 100-105, Thieme, Stuttgart. 3. Porra, R. J., and Grimme, L. H. (1978) Arch. Biochem. Biophys. 164, 3 12-32 1. 4. Meisch, H.-U., and Bellmann, I. (1980) Z. Pfanzenphysiol. 96, 143- 15 1. 5. Mauzerall, D., and Granick, S. (1955) J. Biol. Chem. 219,435-446.

Dalgliesh, C. E. (1952) Biochem. .I. 52, 3-14. Meisch, H.-U., and Bielig, H. J. (1975) Arch. Microbiol. 105, 77-82. 8. Meisch, H.-U., and Bauer, J. (1978) Arch. Microbial.

6. 7.

177,49-52. 9.

Porra, R. J., and Klein, 0. (1981) Anal. Biochem. 166, 51 l-518.

Porra, R. J., and Meisch, H.-U. (1984) Trends Biochem. Sci. 9,95-104. 11. Wolf, U. (1984) Dissertation, University of Saarbriicken. 12. Kissel, H. J., and Heilmeyer, L., Jr. (1969) Biochim. Biophys. Acta 177, 78-87. 10.