An Enzyme Electrode Based on Lipoxygenase Immobilized in Gelatin for Selective Determination of Essential Fatty Acids

Abstract

An enzyme electrode for the specific determination of ω‐3 and ω‐6 fatty acids from the mixture of essential fatty acids (EFAs) was developed by using lipoxygenase (LOX) (EC 1.13.11.12) from soy beans in combination with a dissolved oxygen (DO) probe. The enzyme electrode showed different sensitivities for linoleic (LA) and α‐linolenic acids (ALA), the most common essential fatty acids. Enzyme electrode response depends linearly on LA concentration between 12.8–160.5 µM and ALA concentration between 3.8–18.9 µM in borate buffer, 0.2 M at pH 9.0. However, in phosphate buffer 0.2 M at pH 6.0 linearity is in the range of 7.5–22.5 µM of ALA concentration at 5 minutes response times. Moreover, maximum electrode response was found in borate buffer at pH 9.0 and 30°C.

• Essential fatty acid (EFA)
• Linoleic acid (LA)
• α‐linolenic acid (ALA)
• Biosensor
• Enzyme electrode
• Lipoxygenase (LOX)

Introduction

Essential fatty acids (EFAs) are a group of 12 compounds, but only linoleic acid (LA) and α‐linolenic acid (ALA) are found in diet, particularly in vegetable oils, in some abundance. The other 10 EFAs are either metabolic products of LA and ALA or are found in the diet considerably smaller amounts (Hsu et al., [1998]; Sinclair, [1956]). While an absolute EFA deficiency in the diet might be rare, a lowering of EFA concentrations in the body tissues is common because of the increasing level of other non‐EFA fats in the Western diet. These non‐EFA fats include saturated fats as well as trans‐unsaturated fatty acids which are formed in food processing (Miyashita et al., [1993]).

The steadily growing knowledge about the correlation between the fatty acid composition of the diet and clinical disorders leads to a growing demand for a rapid and easy to use analytical device for fatty acid determination in foods. Up to date the fatty acid composition of fats and oils determined mainly by gas chromatography (GC). Although GC is well established in lipid analysis and offers high sensitivities, it is still time consuming and laborious (Court et al., [1993]; Liminga and Oliw, [2000]).

In this study, we described a biosensor for the determination of ω‐3 and ω‐6 fatty acids from the mixture of EFAs based on LOX from soybeans. LOXs in general catalyse the oxygenation of PUFAs containing cis, cis‐21,4‐pentadiene system by molecular oxygen. The oxygen consumption due to the LOX catalysed oxygenation of EFAs can be monitored amperometricaly by using oxygenmeter (Axelrod et al., [1981]). Since, LA and ALA show differences in first and second oxygenation activity, it is possible to analyse each of them in EFAs mixture. LOX was immobilized directly on DO probe by copolymerization with gelatin, using the bifunctional agent glutaraldehyde as a crosslinker (Ertaş et al., [2000]).

Materials and Methods

Materials

Lipoxygenase (LOX) (EC 1.13.11.12), a lyophilized preparation derived from soy bean, linoleic acid, 225 bloom calf skin gelatin and glutaraldehyde were obtained from sigma Chem.Co. (St. Louis, MO, USA). α‐Linolenic acid was purchased from Fluka Chemie, AG (Switzerland). All other chemicals were of analytical grade.

Apparatus

WTW inoLab Oxi Level 2 model dissolved oxygenmeter based on amperometric made was used for the experiments.

All signals were recorded as dissolved oxygen level(mg/l).

Electrode Preparation

LOX (10 000 IU) and gelatin (20 mg) were mixed at 38°C in 300 µl of phosphate buffer (pH 7.5, 50 mM). Then, 200 µl of the solution spread over the DO probe membrane and allowed to dry at 4°C for 1 hour. Finally, it was immersed in 2.5% glutaraldehyde in 50 mM phosphate buffer (pH 7.5) for 5 min. The enzyme electrode contained 6666 IU cm^{− 2} enzymatic activity.

Measurement of the Oxygen Consumption by the Enzyme Electrode

LOX (linoleat: oxygen oxidoreductase), catalyses the oxygenation of the pairs cis‐double bonds of LA and all other PUFA which contain at least one pair of cis‐double bonds separated by a methylene group located at 8C‐atom. The concentrations of ALA and LA were obtained by measuring the reduction current of oxygen. The steady‐state current depends on the amount of oxygen that is consumed by the enzymatic reaction at the immobilized enzyme membrane which is covered on top of the dissolved oxygen probe. Measurements were performed by standard curves which were obtained by the detection of consumed oxygen level, related to LA and/or ALA concentration in the reaction medium. In the steady state method, addition of substrate caused current decrease due to oxygen consumption in the bioactive layer, which reached steady state in 10 or 5 min depending on both oxygenation type and DO concentrations.

Working Conditions

The measurements of the first oxygenation activity were performed in borate buffer (pH 9.0, 0.2 M) by using both ALA and LA as substrates. The second oxygenation activities were carried out in phosphate buffer (pH 6.0, 0.2 M) by using ALA as a substrate (Schoemaker et al., [1997]). All measurements were carried out using a thermostatic cell at 30°C.

ALA and LA solutions were prepared by mixing of ALA (or LA) and working buffer solution containing Tween‐20 (1%, w/v).

Results and Discussion

Enzyme Electrode Optimization

Effect of pH

The effect of pH at the range of 4–10 on the electrode response was studied with ALA and LA, respectively. Figure 1 showed that the highest response was observed at pH 9.0 for both ALA and LA.

Figure 1. Effect of pH on the electrode response (pH 4.0–5.0: acetate buffer, pH 6.0–8.0: phosphate buffer, pH 9.0–10.0: borate buffer; LA: 64.2 µM, ALA: 7.5 µM, 30°C).

Effect of Temperature

The effect of assay temperature (15–35°C) was examined by using LA. As is shown in Figure 2, maximum sensor response was found at 30°C. Therefore, this value was accepted as optimum temperature for all subsequent experiments.

Figure 2. Effect of temperature on the electrode response (LA: 64.2 µM, in borate buffer, 0.2 M, pH 9.0).

Analytical Characteristics

Linear Range

LOX enzyme electrode response depends linearly on LA concentration between 12.8–160.5 µM (Figure 3) and ALA concentration between 3.8–18.9 µM (Figure 4) at 10 minutes response times in borate buffer (pH 9.0). Linear graphs, defined by the equation y = 0.0018X + 0.0034, (R² = 0.9936) and y = 0.0108X + 0.0062, (R² = 0.9811) were obtained, respectively for LA and ALA. Linearity was found in the range of 7.5–22.5 µM of ALA concentration at 5 minutes response times in phosphate buffer (pH 6.0). At higher concentrations, standard curve showed a deviation from linearity. The deviation was due to insufficient amounts of dissolved oxygen which is a co‐substrate of the enzyme.

Figure 3. Calibration graph for LA (in borate buffer; 0.2 M, pH 9.0, at 30°C).

Figure 4. Calibration graph for ALA (in borate buffer; 0.2 M, pH 9.0, at 30°C).

Reproducibility

The reproducibility of the enzyme electrode was tested by 10 average standard solutions containing equal amounts of LA and ALA, respectively. 63.5 µM of LA and 7.3 µM of ALA were used at borate buffer. The standard deviation (SD) and variation coefficient (CV) were calculated as ± 0.21 µM, 0.33% (n = 10, for LA) and ± 0.11 µM, 1.56% (n = 10, for ALA). Moreover, 13.75 µM of ALA was used at phosphate buffer. The standard deviation (SD) and variation coefficient (CV) were found as ± 0.247 µM and 1.79% (n = 8).

EFA Detection

Total amount of EFAs could be easily detected in previous works (Shafer, [1998]). However, it is important to detect each of them in presence of the others. As defined in equations, LOX electrode showed different sensitivities towards LA and ALA. Soybean LOX isoform I has been reported to act upon PUFA containing suitably positioned all cis‐1,4,7‐octatriene moiety to generate the dihydroperoxy derivatives while consuming 2 mol O₂ per mol substrate. The enzyme prefers the ω‐6 carbon atom of the fatty acid for the first oxygenation (at pH 9.0) (i.e. the sixth carbon atom from the methyl end). LOX may catalyse a second oxygenation if the first oxygenation product still exhibits a cis, cis‐1,4‐pentadiene system as do the oxygenation products of arachidonic acid and α‐linolenic acids. It has been through for a long time that ALA with the first double bond at ω‐3 carbon, is not subject to a second oxygenation step, as the product of the first oxygenation 10‐DHPOT, lacks a cis, cis‐1,4‐pentadiene system. Then, it was reported that soybean LOX is capable of catalyzing multiple oxygenation of ALA, thus generating 9, 16‐DHPOT. If such a multiple oxygenation takes place, any LOX based sensor with an oxygen electrode as a transducer will show a higher sensitivity for ALA (ω‐3 fatty acid) as compared to LA (ω‐6 fatty acid) (Sok and Kim, [1990]). Our results supported these findings. ALA can be detected with higher sensitivity by using our system in comparison to LA.

Moreover, LA was tested in pH 6.0 by LOX electrode but no signal was observed in these conditions. Standard curve for ALA was defined by equation y = 0.0043X‐0.0021, with a correlation coefficient R² = 99.15 (Figure 5). It was also observed that equal amounts of LA and ALA gave the same curve with ALA. And also, Table 1 shows the signals (ΔDO) from the first oxygenation of LA in presence of ALA. These results indicated that we could easily detect EFAs as totally and separately depending on the pH.

Figure 5. Calibration graph for ALA (second oxygenation conditions: in phosphate buffer; 0.2 M, pH 6.0, at 30°C).

Table 1. The Signals (ΔDO) from First Oxygenation of LA in the Presence of ALA

LA conc. (µM)	ΔDO(mg/l) (for LA)	ΔDO(mg/l) (for ALAa)	ΔDO(mg/l) (for LA + ALA)
12.84	0.03	0.04	0.07
32.10	0.07	0.04	0.10
64.2	0.12	0.04	0.16
96.3	0.16	0.04	0.19

^aALA conc: 3.8 µM.

Acknowledgment

This work was supported by Ege University Research Fund Project 97 Fen 008.