Volatile fatty acids of the ribbed mussel, Geukensia demissa

(Smlp. Biochem. Physiol. Vol. 74B, No. 3, pp. 539 to 542. 1983 Printed in Great Britain.

0305-0491,83,,'030539-04503.00,'0 © t983 Pergamon Press Ltd

VOLATILE FATTY A C I D S OF THE RIBBED MUSSEL, G E U K E N S I A D E M I S S A * MING-SHAN HO and PAUL L. ZUBKOFF Department of Environmental Physiology, Virginia Institute of Marine Science, and School of Marine Science, College of William and Mary, Gloucester Point, VA 23062, USA (Received 2I June 1982)

Abstract 1. Volatile fatty acids were extracted and purified from the mussel, Geukensia demissa, by a simple solvent extraction method and quantified by gas liquid chromatography. 2. Propionate was most abundant with about 3/~mol/g dry weight, iso-butyrate and butyrate were near 1 Ftmol/g dry weight; iso-valerate was present in trace amount and valerate was virtually absent. 3. The anaerobic accumulation of propionate is disucssed.

INTRODUCTION Volatile fatty acids (VFA) of invertebrates have not been carefully studied as a group, although one of them, propionate, has been found to be a major end product of anaerobiosis in freshwater snails (Mehlman & von Brand, 1951: von Brand et al., 1955), bivalves (Kluytmans et al., 1975. 1977; G~ide, 1975; GS.de et al., 1975; Ho & Zubkoff, in press), parasitic helminths (de Zoeten et al., 1969; K6hler & Stahel, 1972), lugworms (Surholt, 1977) and earthworms (Gruner & Zebe, 1978). Bivalves and many other invertebrates have high tolerance to anoxia and this ability has been attributed to their anaerobic metabolic pathways which are different from these of vertebrates (de Zwaan, 1977). Volatile fatty acids are conceivable to be important in many other physiological and biochemical processes essential to the survival of bivalves, thus, their analyses are potentially useful. This present paper describes the application of a simple solvent extraction and g a ~ l i q u i d chromatographic method (Gibbs et al., 1973) to isolate, purify and quantify VFA of the ribbed mussel, Geukensia demissa. The function of VFA in anaerobic metabolism of bivalves is also discussed.

extracted and purified from spiked mussel tissue homogenate by procedures based on these of Gibbs et al. (1973) and summarized in Fig. 1. Procedure blanks were processed parallelly with samples to determine the cleanliness of volatile fatty acids extracts. A Hewlett-Packard 7626A ga~liquid chromatograph equipped with flame ionization detectors was employed for this study. The column (glass, 6ft x 1/8 in. i.d.) was packed with 8(F100 mesh Chromosorb WAW coated with 10% SP 1200 supplemented with 1°~ phosphoric acid (Supelco, Inc.). Before a new column could be used successfully, it was conditioned for 24 hr at 150°C; Silyl-8 column conditioner (Pierce Chemical Co.) was injected periodically to restore column efficiency. Temperature programming was from 70 140°C at 4°C/min. Detector response factors were determined from gas liquid chromatograms by injecting a standard mixture of volatile fatty acids. The mixture was prepared by dissolving

TISSUE HOMOGENIZED INTERNALSTANDARDADDED (n-VALER1C ACID) PRECIPITATED (HCIO4) CENTRIFUGED

I

I

PRECIPITATE (DISCARDED)

SUPERNATANT BASIFIED (KOH) CENTRIFUGED

MATERIALS AND METHODS

I

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Ribbed mussels (Geukensia demissa) with shell lengths of 11.5 ± 1.0 cm were collected on the bank of York River near Mumford Island, Virginia. They were apparently healthy and had tissue wet and dry weight of approx. 50 and 3 g, respectively. During the course of this study, salinities were from 14 to 18",,,,. Mussels were cleaned, pried open and then tissues were minced with scissors and homogenized with cold buffer solutions of 2-amino-2-methyl-l-propanol (0.1 M, pH 10. Sigma Chemical Co.) using a VirTis 45 homogenizer IGardmer, NY). 4.38 l~mol of ~l-valeric acid was then added to the tissue homogenate as internal standard; preliminary analyses showed that n-valeric acid was virtually absent in the tissues of G. demissa. Volatile fatty acids were then

ETHYL ACETATE FRACTION AQUEOUSFRACTION VOLATILE FATTY ACIDS (DISCARDED) FOR GLD ANALYSES

* Contribution No. 1064 from the Virginia Institute of Marine Science.

Fig. 1. Preparation of volatile fatty acids from mussels, Geukensia demissa, for gas liquid chromatographic analyses.

SUPERNATANT PRECIPITATE SATURATED (DISCARDED) WITH NoCl EXTRACTEDWITH ETHER

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I

AQUEOUS FRACTION ETHER FRACTION ACLDIFIED (HCI) (DISCARDED) EXTRACTED WITH ETHYL ACETATE

1

539

541)

MING-SHAN Ho and PAUL L. ZUBKOFF

volatile fatty acids. The mixture was prpared by dissolving 100 td each of propionate, iso-butyrate, butyrate, iso-valerate and valerate (Sigma Chemical Co.) in 5 ml of 1 N acetic acid. Chromatograms of VFA were obtained by injecting convenicm volumes (I ;d} of mussel tissue extracts into GLC. Integrated peak area of each volatile fatty acid was then converted to the weight (/~mol) by detector response factor obtained from standard mixtures. A multiplication factor of each chromatogram was determined and which is the ratio of the n-valerate (4.38/~mol) added to the mussel tissue homogenate and the Izmol of n-valerate calculated from thc chromatogram. The multiplication factor was used to convert the wei~,ht of each VFA determined from the chrnmatogram to that of it in the mussel tissue homogenatc. Detail procedures of anaerobic incubation of G. demissa were described in Ho & Zubkoff (in press). Anaerobic condition was created by flushing filtered river water with N 2 and incubation was conducted at 15°C. Both fixed term and time course studies were conducted. For fixed term study. 8 mussels were acclimated in aerated water at 15:C for 2 days and then randomly divided into aerobic and anaerobic sets and incubated for 51 hr. For time course study, 23 mussels were randomly chosen for this experiment. Eight were sacrificed immediately after field collection, three after 2 days aerobic acclimation and the remainmg mussels were incubated anaerobically for periods up to 4 days, with three of them sacrificed daily.

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TEMPERATURE (°C)

RESt LTS Satisfactory extraction, purification and g a > l i q u i d c h r o m a t o g r a p h i c q u a n t i t a t i o n of volatile fatty acids from mussels, Geukensia demissa, has been achieved in this study. Single resolved peaks were o b t a i n e d for acetic, propionic, iso-butyric, n-butyric, iso-valerie and n-valeric acids as s h o w n in Figs 2(AF(C). C o n sistent retention times, baseline resolutions, stable baselines and clean c h r o m a t o g r a m s can be observed. Figures 2(B) and (C) d e m o n s t r a t e V F A extracted a n d purified from mussel tissue h o m o g e n a t e s with the addition o f the internal standard, n-valerate; the a n a e r o bic a c c u m u l a t i o n of p r o p i o n a t e can be observed.

Fig. 2. Gas liquid chromatograms of volatile fatty acids. (A). Reference: standard acid mixture; (B). Geukensm demissa after 51 hr aerobic incubation: (C). Get&ensia demissa after 51 hr anaerobic incubation. C2 - A c e t a t e : C3 - Propionate; iso-C~t - iso-Butyrate; Ca. Butyrate; i s o - C s - iso-Valerate; Cs Valerate (internal standard, n-valerate was predetermined to be absent in tissue extracff of G. demissa.

C o n c e n t r a t i o n s of volatile fatty acids of G. demissa are s u m m a r i z e d in Table 1. Acetic acids could not be d e t e r m i n e d accurately because a peak with similar retention time was detected in p r o c e d u r e blank: con-

Table 1. Volatile fatt~ acids of Geukensia demissa. Anaerobic incubation was under N 2 and at 15 C Volatile Fatty Acids Propionate Fixed T e r m Study

(~mol/g dry weight) Butyrate

iso-Valerat~

(December)

51 hours incubation Aerobic (n=4) A n a e r o b i c (n=4)

Time Course Study

iso-Butyrate

3.5±0.9 14.2±3.2

1.2±0.1 1.2±0.2

I.I±0.I i.I±0.I

0.2±0.0 0.2±0.0

3.3±0.3 2.7±0.6

0.6±0.0 0.5±0.0

0.8±0.1 0.6±0.1

trace trace

2.2±0.2 7.0±1.6 19.8±1.8 33.4±9.0

0.4±0.1 0.5±0.i 0.6±0.1 0.7±0.0

0.6±0.1 0.7+0.1 0.9±0.1 1.0±0.i

trace trace trace trace

(June)

After field collection (n=8) After two days aerobic a c c l i m a t i o n (n=3) A n a e r o b i c i n c u b a t i o n (n=3) Days 1 2 3 4

D a m represent ~ + SE,

Volatile fatty acids of mussels siderable efforts have been attempted to eliminate it but failed'. Within four volatile fatty acids determined, propionate was most abundant (~3/~mol/g dry weight, aerobic condition), followed by iso-butyrate and n-butyrate (~ 1 #mol/g dry weight), iso-butyrate was present only in trace quantity. Propionic acid accumulated appreciably after 51 hr (for fixed term study) and 2 days (for time course study) but not after one day of anaerobic incubation (Table i); this suggests a lag of anaerobic accumulation of propionic acids. Concentrations of iso-butyrate, butyrate and iso-valerate did not change anaerobically. Seasonal variations of VFA contents in G. demissa were not observed. DISCUSSION

The volatile fatty acids of mussels, Geukensia demissa, have been successfully determined as shown in Fig. 2 and Table 1. To facilitate comparison of VFA in G. demissa to those of other bivalves, the concentrations of VFA in G. demissa, M. edulis (Kluytmans et al., 1975), C. edulis (Ggde, 1975) and G0.de et al., 1975) are compiled in Table 2. Although precise comparisons are inappropriate because of variations in experimental conditions and analytical methods, some generalities are nevertheless noteworthy. Anaerobic accumulation of propionate but not iso-butyrate, butyrate, iso-valerate are agreeable in all these studies. Concentrations of volatile fatty acids are also comparable except high butyrate content reported in C. edule (GS.de, 1975). It is not unreasonable to conclude that most bivalves have similar contents of volatile fatty acids. Metabolites analyses have been proved to be useful in investigation of numerous physiological and biochemical phenomena; one good example is the anaerobic accumulation of propionic acid in bivalves dis-

541

cussed in this study. Although pathways for propionate formation in anaerobic tissues have not been carefully investigated in bivalves, they have recently been proposed for parasitic helminths (Tkachuck et al., 1977; Saz et al., 1978). (1) Succinate + Propionyl-CoA ~ Succinyl-CoA + Propionate (2) Succinyl-CoA ~ Methylmalonyl-CoA (3) Methylmalonyl-CoA + E ~ CO2-E + PropionylCoA (4) CO2-E + ADP + H3PO,, ~ CO2 + ATP + E Sum (5) Succinate + ADP + H3PO 4 ~ Propionate + COE + ATP Available evidence seems to suggest that similar pathways for propionate formation also exist in bivalves, thus, the accumulation of propionate lags behind succinate and propionate accumulation concurs with the leveling-off of succinate content during the extended anaerobiosis of G. demissa (Ho & Zubkoff, in press). These pathways for propionate formation have been incorporated in a bivalve anaerobic metabolic scheme proposed by Ho & Zubkoff (in press) to explain the anaerobic accumulation of propionate, succinate and alanine in G. demissa. This scheme contains a modified Embden-Meyerho~ Parnas glycolytic pathways, carbon dioxide fixation, the Krebs cycle, pathways for propionate and alanine formation; it also takes into account of cytosol-mitochondrial compartmentation and attains redox balance overall. REFERENCES

BRANDT. VON,MCMAHONP. & NOLANM. O. (1955) Observations on the postanaerobic metabolism of some fresh-water snails. Physiol. Zool. 28, 35 40.

Table 2. The concentrations (#mol/g dry weight) of volatile fatty acids in several bivalve species Vclatile Fatty Acids Propionate Ribbed Mussel, G e u k e n s i a demissa Aerobic Anaerobic Mussel,

Aerobic Anaerobic

Kluytmans et al.

(3 d)

Common Cockle C a r d i u m edule, Gade Aerobic Anaerobic

(15 h)

(30 h)

iso-Valerate

1.2 1.2

i.i I.i

0.2 0.2

NR NR

i. 0 2.1

ND 0.4

NR NR

33.9 33.4

NR NR

NR NR

NR NR

(1975)

0.9 26.6 (1975) ND 3.1

F r e s h w a t e r Bivalve, A n o d o n t a cygnea, Gade et al. Aerobic Anaerobic

Butyrate

(Table i) 3.5 14.2

(51 h)

Mytilus edulis,

iso-Butyrate

(~mol/g dry weight)

1.6 46.0

(1975) NR NR

Incubations were at 12-I5°C and anaerobic incubation was under N2. ND. not detected. NR, not reported.

542

MING-SHAN HO and PAUL L. ZUBKOFF

GXDI G. (1975) Anaerobic metabolism of the c o m m o n cockle Cardcum edule. I. The utilization of glycogen and accumulation of multiple end products. Archs. int. Physiol. Biochem. g3, 879 886. GS, I)Ii G.. WILPS H., KLt YTMAXSJ. H. F. M. & ZWANN A. DE (1975) Glycogen degradation and end products of anaerobic metabolism ira the fresh water bivalve Anodonta c'y~31wa. J. cutup. Physiol. 104, 79 85. GIBBS B. F.. IIIABA K.. CR,~WIIALL J. C., COOPER B. A. & MAMIR O. A. (1973) A rapid gas chromatographic method for the quantitation of volatile fatty acids in urine. Propionic acid excretion in vitamin B~ 2 deficiency. J. Chromat. 81, 65 69. GRUNER B. & ZEBE E. (1978) Studies on the anaerobic metabolism of earthworms. Comp. Biodwm. Physiol. 60B, 441 445. HD M.-S. & ZUBKOFV P. L. Anaerobic metabolism of the ribbed mussel. GeukeJls'ia demissa. Comp. Bioehem. Physiol. 73, 931 936. KLt'flMAXS J. H.. VIiI!NHOF P. R. & ZWAAN A. DE (1975} Anaerobic production of volatile fatty acids in the sea mussel Mytilus edulis L. J. comp. Physiol. 104, 71 78. KLUYTMANS J. H.. BONI A. M. T. DE, JANUS J. & WIJSMAN T. C. M. (1977) Time dependent changes and tissue speciticities in the accumulation of anaerobic fermentation products in the sea mussel Mytilus ednlis L. Comp. Biochem. Physiol. 58B, 81 87.

KOHLER P. & STAHEL O. F. (1972l Metabolic end products of anaerobic carbohydrate metabolism of Dicrocoelium dendriticum (Trematoda). Comp. Biochem. Physiol. 43B, 733 741. M~HLMAN B. & BRAND T. YON (1951) Further studies on the anaerobic metabolism of some fresh water snails. Biol. Bull. 100, 199 205. SAZ H. J., DUNUAR G. A. & GARm, ERA. E. (1978J Propionate formation from succinate and possible ATP generation. Abstract. The American Society of Parasitologist Meeting. Chicago, Illinois. SURHOLT B. (1977) Production of volatile fatty acids ira thc anaerobic carbohydrate catabolism of Arenieola marina. Comp. Biochem. Physiol. 58B, 147 150. TKACHUK R. D., SAZ H. J., WEINSrklN P. P., FINNtGAN K. & MUELLER J. F. (19771 The presence and possible function of methylmalonyl CoA mutase and propionyl CoA carboxylase in Spirometra mansommles. J. Parasit. 63, 769 774. ZOETEN L. W. DI=, POSTHUMA D. & TIPKIR J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica. Hoppe Seyler's Z. physiol. Chem. 350, 683 690. ZWAAN A. DE (1977} Anaerobic energy metabolism in bivalve molluscs. Oeeamxlr. mar. biol. Ann. Rev. 15, 103 187.