Taurine content of Mexican beans

JOURNAL

OF FOOD

COMPOSITION

AND ANALYSIS

4,322-328

( 199 1)

Taurine Content of Mexican Beans H. PASANTES-MORALES’ Institute

de FisiologLa

Celular,

Universidad

AND R. FLORES National

Autdnoma

de Mkxico,

Mkxico

Received November 1, 1990, and in revised form July 10, I99 1 The taurine content of leguminous seeds,including six varieties of Mexican beans, was examined. Beans (Phaseolus vulgaris) contained taurine at concentrations ranging from 29 to 100 nmol/g. The taurine content of soy bean was 25 nmol/g. Other leguminous seeds containing taurine were lentils (25 nmol/g), chick peas (35 nmol/g), and horse beans (43 nmol/g). About 50% of the taurine content is leached out during soaking. Taurine concentration was not significantly affected by cooking procedures. These results suggest that the concentration of taurine present in beans may be. sufficient to provide the taurine requirements of populations in which the average daily consumption of beans is high. 0 1991 Academic press, Inc.

INTRODUCTION The question of taurine being an essential nutrient in some species has been raised by the following observations: (1) Patients fed a parenteral diet which does not contain taurine present low levels of plasmatic taurine and abnormalities in the electroretinogram (Geggel et al., 1985). (2) Lactant monkeys receiving a formula without taurine show degeneration of photoreceptors, abnormal electroretinogram, and decreases in visual acuity (Sturman et al., 1984). (3) Cats fed a tam-me deficient diet present profound alterations of the structure and function of photoreceptors, characterized by vesiculation and membrane disintegration and a progressive decline of the amplitude of the ERG which is finally undetectable. Eventually cell death occurs resulting in blindness (Hayes et al., 1975; Berson et al., 1976). All these retinal abnormalities are prevented by supplementation with taurine before the occurrence of irreversible damage (Berson et al., 1976). The requirement of taurine for normal brain development is suggested by the observation of delayed and defective maturation and migration of cells in brain cortex and cerebellar cortex of kittens born from taurine deficient mothers (Stumran et al., 1985, 1987) and also in lactant monkeys receiving a tam-me-free formula (Sturman, 1983). All these species in which adverse consequences of taurine deficiency have been observed possess a limited ability to synthesize taurine from endogenous precursors (Jacobsen et al., 1964) and depend to a large extent on dietary intake to maintain endogenous taurine pools including those in maternal milk (Sturman et al., 1987). Taurine is present at high concentrations in muscle (6-40 mM) (Jacobsen and Smith, 1968) and consequently meat is the natural dietary source of the amino acid. In a previous study (Pasantes-Morales et al., 1989) in which we examined the taurine content in a large number of edible parts of vegetables, it was observed that only some seeds and nuts contain detectable amounts of taurine, within a range of 5-50 nmol/ g. Significant differences in concentration were observed in two varieties of beans ’ To whom reprint requests should be addressed. 0889-1575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

322

TAURINE

CONTENT

OF BEANS

323

analyzed and this observation prompted us to examine a larger number of varieties of beans and other leguminous seeds. We also examined the effect of the procedures currently used to prepare these seeds for food consumption. METHODS Preparation of Samples

Seeds were obtained from the local market in 1990 and all came from the seasonal crop of the year. Normally seeds were stored in the laboratory at room temperature for no more than 2 weeks. For amino acid analysis, seeds were weighed, ground to obtain a flour, and 50-500 mg of this flour was extracted with 70% ethanol. To determine the optimum extraction conditions, seed to ethanol ratios (w/v) varied from I/ 10 to l/50. The ethanol extracts were centrifuged at 900 g for 10 min and the pellets were washed twice. The combined supernatants were treated following the procedure of Awapara ( 1948) to obtain an extract free of lipids. The procedure consists in mixing aliquots of 1.5 ml of the ethanol extract with 4 ml of chloroform, followed by vigorous stirring in a Vortex for 3 min and centrifugation at 900 g for 10 min at room temperature. The water phase free of lipids and containing the extract of amino acids was separated with a Pasteur pipet. The pH of the extracts was adjusted to 1.O, and then samples were passed through a cation exchange column (AG 5OW-X8, 100-200 mesh, H+ form, 5 X 70 mm) and eluted with water (3 ml). By this procedure, 85% of taurine is eluted from the column while most amino acids were retained (Pasantes-Morales et al., 1989). This cleaning procedure eliminates a large number of amino acids present in seeds and allows samples eluted from the column to be run at higher sensitivity. Some samples were hydrolyzed with HCl in an attempt to eliminate acidic compounds that might interfere with taurine analysis. No differences were found in the taurine content in the hydrolyzed and nonhydrolized samples. Recovery studies were done by adding known amounts of 3H-labeled taurine and unlabeled taurine to some samples. The samples were processed as described above. Eighty percent of the added taurine was recovered. Chromatographic

Procedure

The taurine content in protein-free, lipid-free extracts of seeds was analyzed by reversed-phase, high performance liquid chromatography (HPLC). The gradient consisted in methanol/potassium acetate as described by Geddes and Wood (1984). Ophtaldialdehyde derivatized amino acids were prepared by mixing the extracts with 0-phtaldialdehyde, 1 mg/ml in 0.4 M borate, pH 10.4, for exactly 3 min. The detection limit of the chromatographic method was 20 pmol. The detector response was linear (? < 0.99 in the range of 20-200 pmol). The average coefficient of variability attributable to the HPLC assay procedure was 0.92% as determined by analysis of 12 replicate injections of working standards. Sample extracts were spiked with a taurine standard to determine the simultaneous appearance on the chromatogram of the sample and standard taurine peaks. To examine the effect of cooking on the taurine content, the original sample (one ground seed) was divided into two equal portions to avoid differences due to individual variability among seeds. Amino acids were extracted from one portion as described above and the other portion was boiled for 60 min in a known volume of water. The

324

PASANTES-MORALES

AND

FLORES

taurine content of the cooked portion and of the boiling fluid was then assayed. The final taurine values are expressed as nanomoles per gram. RESULTS AND

DISCUSSION

The taurine content of six varieties of beans (Phase&~) was examined. Four other species of leguminous seeds were also analyzed. Taurine concentration in all varieties of beans ranged from 30 to 100 nmol/g. The lowest concentration was found in the white bean (Alubia) and the highest in the variety Garbancillo. The other leguminous seeds examined (horse bean, chick pea, soy bean, and lentil) had similar ranges of taurine concentration, the soy bean had the lowest taurine content (Table 1). The variation of taurine concentration among individual seeds was high and therefore a large number of individual seeds had to be analyzed in order to obtain representative means. In contrast, the coefficient of variability of the assays of single seeds was low, commonly not exceeding 2% (Table 1). Somewhat lower concentrations of taurine in seeds have been reported earlier (Pasantes-Morales et al., 1989; Grosvenor et al., 1987). The differences may be due to the choice of procedure for extraction of taurine. In the present study of the optimal extraction conditions, increasing volumes of ethanol were used for extraction to give ethanol-to-ground seed ratios (v/w) ranging from l/ 10 to l/50. The extraction efficiency was inversely related to this ratio up to a ratio of l/30 as is shown in Fig. 1. The optimal extraction conditions were not the same for all the varieties of seeds. Most of them showed an increase in the efficiency of extraction related to ethanol volume similar to that of the Garbancillo variety illustrated in Fig. 1. However, in others such as in the chick pea, the increase in taurine content was only twofold at an extraction ratio of l/50 as compared to a l/ 10 ratio, whereas,

TABLE TAURINE

CONCENTRATION

1

OF BEANS AND OTHER

Seed

Taurine

CV

n

46 100

5.8

0.38 0.29 0.52 0.61

44 40 25 25

0.41

25

43 25 35 25

escttlenta)

seeds were weighed

extraction

and chromatographic

are expressed variability

in nmol/g.

9.9 6.1 4.9 5.1 4.8 6.8 5.3 7.1 4.5

30

Soy bean (Glyche mar) Chick pea (Cicera reikmm)

aGround

Concentration

SE

41 39 35

0.32

25

0.51

25

0.31 0.49 0.38

22 25 20

and used for analysis as described

Samples were analyzed in triplicates

SEEDS’

nmol/g

Bean (Plmeolus uulgaris) Var. Negro Garbancillo Bayo Rosita Flor de mayo Alubia Horse bean (Vicia faba)

Lentil (Lens

LEGUMINOUS

and values obtained analysis

SE Standard

efficiency error,

of each variety or species. n Number

in Methods.

were corrected

(See Methods). CV

Mean

coefficient

of samples analyzed.

for

Results of

TAURINE

CONTENT

l/40

1120 Seed/Solvent

325

OF BEANS

Ratio

l/60 (w/v)

FIG. 1. Taurine concentration in beans (Phuseolus vulgaris, Var. Garbancillo) extracted with different volumes of 70% ethanol. Seeds were ground, weighed, and homogenized with an ethanol volume necessary to give the ethanol-to-seed ratios indicated in the figure. After extraction, the concentration of taurine was measured as described under Methods. Each point in the curve is the mean + SEM of analysis in eight seeds.

in Alubia, a fourfold increase was observed at an extraction ratio of l/50 as compared to the l/ 10 extraction ratio. According to these results, all analyses to determine the concentration of taurine in seeds were carried out using a seed-to-ethanol extraction ratio of l/50. Beans are hard seeds and normally require presoaking and prolonged boiling to be consumed as food. The effect of these procedures on taurine content of beans was examined. Table 2 shows that 40-50% of the taurine content in the dry seed was found in the soaked water. For the Negro bean, which has a concentration of 46 nmol/ g, 18 nmol/g were found in the soaking water, i.e., 39%. For the Garbancillo bean, with a concentration of 98 nmol/g, 53 nmol/g were recovered in the soaking water,

TABLE 2 LEACHING

Seed

OF TAUR~NE

Taurine

FROM BEANS BY PRESOAKING~

content

Taurine

content

of soaked

seeds

of soaking

water

nmol/g

SE

nmol/g

SE

n

Bean (Negro)

26

4.1

18

3.9

(6)

Bean (Garbancillo)

48

5.9

53

7.9

(6)

aSeeds were weighed and presoaked for 12 h in a known volume of tap water. After this time, the sample was homogenized and assayed for taurine as described under Methods. An ahquot of the soaked water was taken for taurine analysis. Results are means + SE of the number of seeds indicated in parentheses.

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PASANTES-MORALES

AND

FLORES

i.e., 54% (Table 2). Boiling seeds for 1 h increased taurine concentration by about lo%, from 98 nmol/g in the uncooked seed to 104 nmol/g in the cooked seed plus the cooking liquid (Table 3). Contradictory results have been obtained regarding the effect of boiling on taurine content of food. Roe and Weston ( 1965) found substantial decreases in the taurine content of meat whereas an increase was observed in oysters after the cooking procedure. We included a standard taurine solution as a control in all the experimental procedures and, as expected, no changes were observed in the concentration of the sample indicating that changes in taurine concentration cannot be due to modifications in the molecule. The increase in taurine content by boiling may be due either to better conditions for extraction in the cooked seed or to an increase in free taurine subsequent to release from chemical combinations destroyed by the boiling procedure. The first possibility seems more likely since, according to the results of the present study, higher ratios of solvent-tissue than those commonly used for amino acid analysis in animal tissues are required to ensure optimal extraction of taurine from seeds. It is unknown whether this requirement is particular of hard seeds or whether it is also a condition to extract all taurine in other edible plant parts. If this is the case, the concentration of taurine actually present in food of plant origin could be significantly higher than that considered at present. Another dietary source of taurine from plants may be that originating from the sulfur amino acids methionine and cysteine present in plant tissues. In animal tissues taurine is synthesized from cysteine via a reaction catalyzed by the cysteine sulfinate decarboxylase, forming hypotaurine which is subsequently oxidized to taurine. The activity of the cysteine sulfinate decarboxylase, which is the enzyme regulating the pathway, shows large variations in the different tissues and species and it is very low in human tissues (Jacobsen et al., 1964; Warden and Stipanuk, 1985). Seeds contain TABLE LEACHING

OF TAURINE

FROM BEANS BY COOKING

Seed

Taurine

Uncooked

nmol/g

Bean

(Garbancillo)

volume

homogenized

SE

nmol/g

SE

nmolig

SE

65

9.1

39

6.3

in Methods

of tap

water

and the taurine

liquid were assayed.

Cooked

of 2 g was divided

as described

Results

Content

Samples

12.5

96

aAn original sample analyzed known

3

Samples

in two portions. and

are means

Liquid

One portion was then

the other portion was boiled in a

for 66 min. The cooked content

Cooking

of this sample + SE of 5 seeds.

portion

was then

and of the cooking

TAURINE

CONTENT

OF BEANS

327

large amounts of the precursor sulfur amino acids (Orr and Watt, 1957) but due to the low activity of the cysteine sulfinate decarboxylase in humans, it is unlikely that these amino acids represent a significant source of taurine to the human diet. No information is available so far about the activity of the cysteine sulfinate decarboxylase in plants or of the contribution of other pathways to the synthesis of taurine in plant tissues. The taurine content of beans and other leguminous seeds found in this study was 60-200 times lower than that found in meat (Jacobsen and Smith, 1968; PasantesMorales et al., 1989). However, this amount may be sufficient to meet the requirements of dietary taurine. Although the daily requirement of the amino acid has not been determined, indirect and approximate estimates may be obtained from the balance between taurine intake and taurine excretion. Values of taurine concentration in urine show large variations (903 ? 580 pmol/day) (Laidlaw et al., 1988) but even considering the lowest values, they are close to the amount of taurine found in a regular red meat portion of 100 g (about 6 pmol/g). These calculations suggest that meat provides an amount of taurine much larger than that which seems necessary for normal requirements. In support of this interpretation is the finding that taurine excretion in urine of vegans is still relatively high (266 f 279 pmol/day) (Laidlaw et al., 1988). In a previous study (Pasantes-Morales et al., 1989) it was found that nuts contain taurine at concentrations similar to those present in beans. However, the lower amounts of nuts currently included in the diet make beans a comparatively more important dietary constituent relevant to taurine intake. Individuals who consume beans as a main component of their diet typically consume about 250 g per day. The levels of taurine found in 250 g of beans may be sufficient to meet the requirements for tam-me. According to the present results, it would be of interest to emphasize the importance of including beans in lactant women in communities in which these seeds are main dietary constituents in order to ensure an appropriate exogenous taurine supply to infants via maternal milk. There is a tendency to include special foods during lactation which might replace the normal supply of beans with potential adverse effects on the taurine content of milk. ACKNOWLEDGMENTS This work was supported by grants from the Stiftung Volkswagenwerk and from the Programa de Apoyo a Proyectos de Investigacibn y de Innovacibn Docente IN-204589. We gratefully acknowledge the support of Professor Bernd Hamprecht who acted as the German partner in the “Program of Partnerships” of the Stiftung Volkswagenwerk.

REFERENCES AWAPARA, J. (1948). Application of paper chromatography to the estimation of free amino acids in tissues. Arch. Biochem. 19, 172-190. BERSON, E. L., HAYES, K. C., RABIN, A. R., SCHMIDT, S. Y., AND GAIL, W. (1976). Retinal degeneration in cats fed casein. II. Supplementation with methionine, cysteine, or taurine. Invest. Ophthalmol. Vis. Sci. 15, 52-58. GEDDES,J. W., AND WOOD, J. D. (1984). Changes in the amino acid content of nerve endings (synaptosomes) induced by drugs that alter the metabolism of glutamate and -aminobutyric acid. .I Neurochem. 42, 16GEGGEL, H. S., AMENT, M. E., HECKENLIVELY, J. R., MARTIN, D. A., MARTIN, D. S., AND KOPPLE, J. D. ( 1985). Nutritional requirements for taurine in patients receiving long term parenteral nutrition. N. England J. Med. 312. 142-146.

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PASANTES-MORALES

AND FLORES

GROSVENOR, H. B., LAIDLAW, S. A., AND KOPPLE, J. D. (1987). Taurine content of common foodstuffs. Fed. Proc., 46-891. HAYES, K. D., CAREY, R. E., AND SCHMIDT, S. Y. (1975). Retinal degeneration associated with taurine deficiency in the cats. Science 188, 949-95 1. JACOBSEN,J. G., THOMAS, L. L., AND SMITH, L. H. (1964). Properties and distribution of mammalian Lcystein sulfinate carboxylases. Biochim. Biophys. Acta. 85, 103. JACOBSEN,J. G., AND SMITH, C. H. (1968). Biochemistry and physiology of taurine and taurine derivatives. Physiol. Rev. 48,424-5 11. LAIDLAW, S. A., SHULTZ, T. D., CECHINA, J. T., AND KOPPLE, J. (1988). Plasma and urine taurine in vegans. Am J. Clin. Nutr. 47, 660-663. ORR, M. L., AND WATT, B. K. (1957). Amino Acid Content ofFoods. Home Economics Research Report No. 4, Agricultural Research Service, U.S. Department of Agriculture, Washington, DC. PASANTES-MORALES,H., QUESADA, O., ALCOCER, L., AND SANCHEZOLEA, R. (1989). Taurine content in foods. Nutr. Rep. Int. 40, 793-801. ROE, D. A., AND WESTON, M. 0. (1965). Potential significance of free taurine in the diet. Nature 205,287288. STURMAN, J. A. (1983). Taurine in nutrition research. In Surfur Amino Acids Biochemical and Clinical Aspects (K. Kuriyama, R. J. Huxtable, and H. Iwata, Eds.), pp. 28 l-295. Liss, New York. STURMAN, J. A., WEN, G. Y., WISNIEWSKI, H. M., AND NEURINGER, M. 0. (1984). Retinal degeneration in primates raised on a synthetic human infant formula. Znt. J. Dev. Neurosci. 2, 12 1- 126. STURMAN, J. A., MORETZ, R. C., FRENCH, J. H., AND WISNIEWKI, H. M. (1985). Taurine deficiency in developing cat: Persistence of the cerebellar external granule cell layer. J. Neurosci. Res. 13, 405-416. STURMAN, J. A., PALACKAL, T., IMAKI, H., MORETZ, R. C., FRENCH, J., AND WISNIEWSKI, H. M. (1987). Nutritional taurine deficiency and feline pregnancy and outcome. In The Biology of Taurine (R. J. Huxtable, F. Franconi, and A. Giotti, Eds.), pp. 113-124. Plenum, New York. WARDEN, J. A., AND STIPANUK, M. H. (1985). A comparison by species, age and sex of cysteinesulfinate decarboxylase activity and taurine concentration in liver and brain of animals. Comp. Biochem. Physiol. B 82, 233-239.