Fluorometric Analysis of 2-Thiobarbituric Acid Reactive Substances in Turkey

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Fluorometric Analysis of 2-Thiobarbituric Acid
Reactive Substances in Turkey1
C. JO and D. U. AHN2
Department of Animal Science, Iowa State University, Ames, Iowa 50011-3150
ABSTRACT
Three experiments were conducted to
develop a sensitive and reliable fluorometric thiobarbituric acid (TBA) reactive substances (TBARS) method to
determine lipid oxidation products in meat. The first
study was conducted to find the optimum sample
preparation conditions for meat in the fluorometric
method. The second study was to compare the original
and the modified methods by using meat and blood
samples. The modified fluorometric method was compared with a conventional spectrophotometric method
in a third study.
Four different extraction solutions (2.5 M acetic acid,
0.5 M hydrochloric acid, 0.8 M perchloric acid, and 1.4
M trichloroacetic acid) and two ratios of extraction
solution to TBA (20 mM) solutions (2:1 and 1:1) were
examined in the first study. Hydrochloric acid was the
optimum among the four extraction solutions tested,
and the ratio of extraction solution to TBA solution at 1:
1 was the best for the fluorometric TBARS method in
raw ground turkey. The modified fluorometric method
had high recovery rates (91%, average), and the
regression coefficient of the standard curve prepared
with spikes was also high (r2 = 0.99).
The analysis of raw meat and plasma samples
indicated that the modified fluorometric method had
greater sensitivity than the original method. The pH of
the reaction mixture played an important role in
extraction TBARS from samples, and low pH conditions
were preferable to high pH conditions. The amount of
lipid oxidation products in raw turkey breast meat
indicated that the fluorometric method had much
greater sensitivity than the spectrophotometric method.
The results from the three studies showed that the
modified fluorometric TBARS method was useful for the
samples with low lipid oxidation products, such as fresh
raw meat. The sensitivity of the modified fluorometric
method also facilitates the determination of oxidation
products in small quantities of samples.
(Key words: fluorometric analysis, spectrophotometric analysis, thiobarbituric acid
reactive substances, lipid oxidation, pH)
1998 Poultry Science 77:475–480
However, Kosugi et al. (1989) reported that the
absorbance of red pigments in TBA methods may not
specifically quantify MDA because other substances may
also react with TBA and contribute to absorbance. In
addition, heating and acidic condition may cause
overestimation of thiobarbituric acid reactive substances
(TBARS). Gray and Monahan (1992) reported that MDA
interacted with various food constituents such as amino
acids, proteins, glycogen, and other food ingredients.
The interaction rendered the MDA unavailable to react
with TBA reagent and resulted in an underestimation of
TBARS.
Because of insufficient specificity and sensitivity of
conventional TBARS methods, alternative analytical
approaches have been developed. Kakuda et al. (1981)
developed a HPLC method to assess the amount of
MDA in aqueous distillates of chicken meat. They found
INTRODUCTION
Lipid oxidation is one of the major causes of quality
deterioration in meat. Several methods have been
developed to assess lipid oxidation products in muscle
foods. The thiobarbituric acid (TBA) test (Tarladgis et al.,
1960) is among the most widely used to quantify lipid
oxidation products in meat and meat products because it
is simple and fast. The TBA test determines the amount
of malondialdehyde (MDA), a major secondary byproduct of lipid oxidation, in a sample. The determination of MDA in meat or meat products has been studied
widely (Hoyland and Taylor, 1991), and significant
correlations between the TBA values and sensory scores
of poultry meat have been reported (Salih et al., 1987).
Received for publication March 28, 1997.
Accepted for publication November 1, 1997.
1Journal Paper Number J-17326 of the Iowa Agriculture and Home
Economics Experiment Station, Ames, Iowa, Project Number 3190, and
supported by Food Safety Consortium and Hatch Act.
2To whom correspondence should be addressed:
duahn@iastate.edu
Abbreviation Key: BHT = butylated hydroxytoluene; DDW =
deionized distilled water; MDA = malondialdehyde; TBA = thiobarbituric acid; TBARS = thiobarbituric acid reactive substances; TCA =
trichloroacetic acid; TMP = 1,1,3,3-tetramethoxypropane.
475
476
JO AND AHN
that HPLC was more sensitive than spectrophotometric
methods in assessing lipid oxidation products in aqueous distillates of chicken. Other secondary products of
lipid oxidation, such as pentanal (Ang and Young, 1989)
or hexanal (Shahidi and Pegg, 1994), also has been used
to determine lipid oxidation by a HPLC, but conventional spectrophotometric TBARS methods are still
preferred over chromatographic methods because of
their simplicity. Yagi (1987) developed a fluorometric
method that can determine lipid oxidation products in
serum and plasma. Yagi’s fluorometric method had
greater sensitivity than the spectrophotometric method,
but was not appropriate for meat.
The objective of present work was to develop a
sensitive, simple, and reliable fluorometric method that
can determine lipid oxidation products in meat. To
accomplish this objective, the following studies were
conducted: 1) the determination of the best sample
preparation conditions for meat, 2) the comparison
between the Yagi (1987) original and the modified
methods using meat and plasma, and 3) the comparison
between modified fluorometric and conventional spectrophotometric methods using meat.
MATERIALS AND METHODS
Chemicals and Reagents
Acetic acid, HCl, trichloroacetic acid (TCA), perchloric
acid (70%), n-butanol, pyridine, ethanol, and SDS were
obtained from Fisher.3 Thiobarbituric acid, butylated
hydroxytoluene (BHT), and 1,1,3,3-tetramethoxypropane
(TMP) were purchased from Sigma.4
Acetic acid (2.5 M), HCl (0.5 M), perchloric acid (0.8 M),
TCA (1.4 M), and SDS (8.1%) were prepared by dissolving
an appropriate amount of each chemical in deionized
distilled water (DDW). The TBA-TCA solution was
prepared by dissolving 20 mM TBA in 15% TCA solution
and BHT (7.2%) was dissolved in ethanol (95%). The nbutanol-pyridine solution (15:1, vol/vol) was made by
mixing 900 mL of n-butanol with 60 mL of pyridine. All
chemicals and reagents were reagent grades and used
without further purification.
Sample Preparation and Storage
Turkey breast meat, ground twice through a
3-mm plate, was used to prepare patties (approximately
100 g). Patties (experimental unit) were stored in a 4 C
refrigerator for 7 d, and analyzed by using the spectrophotometric and the fluorometric methods. Meat (3 or
1 g) was weighed into a test tube (50 mL) with 9 mL of
3Fisher Scientific, Pittsburgh, PA 15219-4785.
4Sigma Chemical Co., St. Louis, MO 63178-9916.
5Brinkman Instruments Inc., Westbury, NY 11590-0207.
6Gilford Instrument Laboratories, Inc., Oberlin, OH 44074.
7Barnstead/Thermolyne Corp., Dubuque, IA 52004-0797.
DDW and homogenized with a Brinkman polytron5 for 15
s at high speed. BHT (7.2%, 50 mL) was added before
homogenization. The homogenate was used for both the
spectrophotometric (3 g) and the fluorometric methods (1
g). Blood samples were taken from live turkeys 1 d before
slaughter. Blood was collected in a test tube containing
anti-coagulant (heparin, 200 unit/mL) and centrifuged for
10 min at 1,000 × g. The plasma was collected and stored in
a freezer (–20 C) until used.
TBARS Analysis
Spectrophotometric Method. The meat homogenate
(1 mL) prepared as above was taken into a disposable test
tube (10 mL), and 2 mL of the TBA-TCA solution were
added. The mixture was vortexed, heated in a 90 C
waterbath for 15 min, cooled in a cold water bath for 10
min, and centrifuged at 2,000 × g for 15 min. The
absorbance of supernatant was measured at 531 nm with a
microsample spectrophotometer.6
Fluorometric Method. Yagi’s original method (1987)
was used except that the samples were measured by a
fluorometer7 (Model 450, Barnstead/Thermolyne) with
520 nm excitation and 550 nm emission instead of 515 nm
excitation and 553 nm emission, due to instrumental
limitations. Four extraction solutions, 2.5 M acetic acid, 0.5
M HCl, 0.8 M perchloric acid, and 1.4 M TCA, were
prepared. To compare the role of TBA reagent on the
yields of TBARS, two extraction solution to TBA solution
ratios (2:1, 1:1, vol/vol) were also used. After selection of
the best conditions for meat samples, the selected
conditions were compared with the original conditions
using plasma and meat samples.
To examine the effect of pH on the yield of lipid
oxidation products, Yagi’s original method (1987) was
used with some modification. Hydrochloric acid (2 M)
was used to adjust appropriate pH conditions for reaction
mixtures before heating, TMP was used as a TBARS
standard.
For the modified method, 0.5 mL of meat homogenate
or 0.5 mL of plasma sample, 200 mL of 8.1% SDS, 1.5 mL of
HCl, 1.5 mL of 20 mM TBA, 50 mL of BHT, and 250 mL of
DDW were added to a test tube. The sample was vortexed
and heated in a 90 C waterbath for 15 min. After cooling
for 10 min, 1 mL of DDW and 5 mL of n-butanol-pyridine
solution was added. The sample was mixed thoroughly,
and centrifuged at 3,000 × g for 15 min. The fluorescence
reading of upper layer was measured by a fluorometer
with 520-nm excitation and 550-nm emission. Measurements were sequentially made at gain 1 and 5.
Statistical Analysis
A two-way ANOVA using the General Linear Models
procedure of SAS (SAS Institute, 1989), was performed
to determine the effect of extraction solution and
extraction solution to TBA reagent ratio on the yields of
TBARS. Three replications were made and four readings
477
FLUOROMETRIC TBARS ANALYSIS
TABLE 1. Fluorescence reading of meat samples with different extraction solutions and
extraction solution to thiobarbituric acid (TBA) (20 mM) reagent combinations1
Extraction solution5
Ratio
Acetic acid
HCl
1:1
2:1
SEM4
Mean
SEM5
44.25cx
35.92cy
1.82
40.08d
63.83ax
57.17ay
2.15
60.50a
Perchloric acid
51.17bx
46.33by
1.44
48.75c
1.64
Trichloroacetic
acid
58.17ax
51.42by
1.09
54.79b
SEM2
Mean
SEM3
2.35
1.97
54.35x
47.70y
1.161
1.161
a–dMeans
within a row with no common superscript differ significantly (P < 0.05); n = 16.
within a column with no common superscript differ significantly (P < 0.05); n = 4.
12.5 M acetic acid, 0.5 M hydrochloric acid, 0.8 M perchloric acid, 1.4 M trichloroacetic acid.
2SEM: Among the means of different extraction solutions with same ratios; n = 12.
3SEM: Among the means of two ratios; n = 24.
4SEM: Among the means of the same extraction solution with different ratios; n = 6.
5SEM: AMong the means of the same extraction solutions; n = 24.
x,yMeans
were made for each replication. Differences among mean
values of fluorescence readings were determined by
calculating Student-Newman-Keul’s multiple range test.
An ANOVA and Student’s t test were performed to
determine differences between the original and modified
methods. A two-way ANOVA was used to determine the
effect of methods and storage time on TBARS values of
meat. Each treatment combination had four replications.
Mean values and SEM were reported.
RESULTS AND DISCUSSION
Sample Preparation Conditions
The fluorescence readings of samples were significantly higher when the ratio of extraction solutions to TBA
solution was 1:1 rather than 2:1 (Table 1). Therefore, the
extraction solution to TBA (20 mM) solution ratio of 1:1
was recommended for extraction the TBA reactive
substances (TBARS) in samples. Among the four extraction solutions, HCl produced the greatest fluorescence
reading in both the ratios of extraction solution to TBA
solution at 1:1 and 2:1. When the ratio of extraction
solution to TBA reagent was 1:1, however, the yields from
HCl and TCA were not significantly different. Tarladgis et
al. (1960) originally used HCl as an extraction solution for
TBARS. Recently, Rosmini et al. (1995) reported that 10%
TCA solution produced the best recovery percentages for
“paté” among combinations of phosphoric acid and TCA.
The fluorescence yields from different extraction solutions
increased in the order of acetic acid < perchloric acid <
TCA < HCl when the fluorescence readings at 1:1 and 2:1
were pooled. However, the fluorescence yields of perchloric acid and TCA were not different when the ratio of
extraction solutions to TBA solution was 2:1, and those of
the TCA and HCl when the ratio was 1:1. Therefore, HCl
would be the acid of choice when the ratio of extraction
solutions to TBA solution is high (2:1) but both TCA and
HCl can be used when the ratio is low (1:1).
The top two extraction solutions, HCl and TCA, were
selected from the yield study, and sequentially used to test
the recovery rates of spikes in meat samples. The HCl had
a greater slope than that of the TCA (Figure 1) and the
average recovery rates of spikes were 91.0 and 64.6%,
respectively (P < 0.01). Therefore, using HCl as an
extraction solution produced 1.5 times higher yield than
that of the TCA. Although both spikes had high regression
coefficients (r2 = 0.99), HCl would be a better extraction
solution for fluorometric analysis than TCA.
Williams et al. (1983) suggested that TBA methods
should report results in terms of their respective standards. Other researchers (Crackel et al., 1988) recom-
FIGURE 1. The influence of extraction solutions on the slopes of
standard curves prepared by the modified fluorometric method. Either
HCl (0.5 M) or trichloroacetic acid (TCA) (1.4 M) was used as an
extraction solution (♦: with HCl, y = 97.7x – 21.4, R2 = 0.998; x: with TCA,
y = 63.4x – 16.2, R2 = 0.996).
478
JO AND AHN
TABLE 2. The amount of lipid oxidation products in meat and plasma samples
determined by the original and modified fluorometric methods
Method
Sample
Meat
Blood
Actual readings from fluorometer
Calculated concentration, mg MDA2/kg meat
Actual readings from fluorometer
Calculated concentration, nmol MDA/mL plasma
Original1
Modified
SEM
23.75b
0.69a
21.00b
0.88a
57.25a
0.52b
32.00a
0.41b
1.07
0.02
0.87
0.03
a,bMeans
(n = 4) within a row with no common superscript differ significantly (P < 0.05).
fluorometric method for serum and plasma (Yagi, 1987).
2MDA = malondialdehyde.
1Yagi’s
mended determination of the recovery values for MDA
and preparation of a standard curve for the calculation of
an appropriate TBA conversion. Therefore, using extraction solutions with higher recovery rates would provide
more accurate and reliable TBARS values than those with
lower rates.
Original vs the Modified
Fluorometric Methods
The modified method had higher readings than those
of the original method (Table 2); however, when the
amount of MDA was converted to TBARS values by using
a standard curve, the modified method had lower values
than those of the original method. The explanation of this
result was revealed to be related to the pH of the two
reaction mixtures prepared from the two methods.
In the Yagi (1987) original method, the pH of reaction
mixture was around 2.0, and that of the standard was 2.7.
Yagi’s original method uses phosphotungstic acid and
sulfuric acid to extract TBARS in blood or serum samples.
The two acids added are removed from the sample after
centrifugation, although a small amount of acids still
remains in precipitants and maintains the pH of precipitants at around 2.0. Phosphotungstic acid and sulfuric acid
are not added when a standard curve is prepared.
Therefore, the pH of the reaction mixture before butanol
extraction is higher (pH 2.7) than that of the sample. In the
modified method, however, acid was added to both
standard and sample preparations and resulted in the pH
of the reaction mixtures being around 1.0.
Figure 2 shows the effect of pH on the yield of lipid
oxidation products determined by the original fluorometric method. The lower the pH and the higher the
fluorescence readings were observed, at the pH range of
0.7 to 2.8. These data indicated that the pH difference in
the reaction mixtures of the modified and the original
methods can cause 1.5- to 2-fold differences in fluorometric readings. The pH effect in standard curves explains the
opposite results between the fluorescence readings and
the calculated amounts of MDA. The calculated values
from the standard curve may produce erroneous values in
Yagi’s method unless the pH differences between the
standard and samples are considered. Therefore, the
calculated amount of MDA from standard curve of
modified fluorometric method is reliable because of the
pH consistency.
Kwon et al. (1965) reported that it is difficult to
hydrolyze all MDA bound to meat proteins without using
strong acidic conditions and heating. Chen and
Waimaleongora-Ek (1981) reported that the pH values
influenced the lipid oxidation of ground raw chicken meat
as measured by a TBA test. They concluded that the lower
the pH values in the sample, the higher were the TBARS
values in the pH range of 3.09 to 9.50. Draper et al. (1986),
on the other hand, suggested that, if strong acid was used,
MDA-TBA complexes would become unstable and cause
problems related to the quantitative recovery of MDA
during a TBA test. The average recovery rate of our
modified fluorometric method, however, was 91% at the
pH range of 0.7 to 2.7 and was consistent. The pH of
reaction mixture in both standard curve and real samples
from modified fluorometric method was 1.0, and were
consistent within the pH range (0.7 to 2.7) that we studied
for pH effect on yields.
FIGURE 2. Effect of pH on the recovery of standards added in meat
samples (y = –66.4x + 385.6, R2 = 0.933).
479
FLUOROMETRIC TBARS ANALYSIS
TABLE 3. The amount of lipid oxidation products of raw turkey breast meat by the
fluorometric and the spectrophotometric methods during the 7-d storage
Storage time
Method
0 d
1 d
2 d
Fluorometric
Spectrometric
SEM3
0.21ex
0.22abx
0.01
0.28dx
0.14cy
0.01
0.38bcx
0.21cy
0.01
3 d
4 d
(mg MDA2/kg meat)
0.33cdx
0.41bx
0.12cy
0.22aby
0.01
0.02
5 d
6 d
7 d
SEM1
0.43bc
0.22aby
0.02
0.50ax
0.18by
0.02
0.55ax
0.25ay
0.03
0.01
0.01
a–eMeans
within a row with no common superscript differ significantly (P < 0.05). n = 16.
within a column with no common superscript differ significantly (P < 0.05). n = 16.
1SEM: Among means of same method with different storage. n = 128.
2MDA = malondialdehyde.
3SEM: Among means of same day with different methods. n = 32.
x,yMeans
The lipid peroxide levels in normal human serum
ranged from 1.86 to 3.94 nmol MDA/mL serum (Yagi,
1987). Our calculated TBARS values of the blood from
17-wk-old turkeys by Yagi’s original method were 0.88
nmol MDA/mL plasma and were 0.41 nmol MDA/mL
plasma by the modified method. The modified method
required less than half the time of the original method, and
the results already discussed indicated that the modified
method is superior to the original fluorometric TBARS for
meat samples.
Modified Fluorometric vs Conventional
Spectrophotometric TBARS Methods
The comparison of the lipid oxidation products in raw
turkey breast meat indicated that the fluorometric method
had a greater sensitivity than the spectrophotometric
method (Table 3). The TBARS values from the fluorometric method gradually increased from 0.21 to 0.55. The
results from the fluorometric method showed significant
changes in TBARS values over storage, but TBARS values
in the same meat samples obtained from the spectrophotometric method were not consistent. No meaningful
differences in TBARS values could be detected during the
7-d storage.
Lipid oxidation product in raw meat is generally very
small because the conditions in raw meat are unfavorable
for the further degradation of the primary oxidation
products to the secondary products. Therefore, it is
difficult to find differences in lipid oxidation products in
raw meat when analyzed by the spectrophotometric
method (Raharjo and Sofos, 1993). Siu and Draper (1978)
investigated MDA content in various raw or cooked
meats. Their TBA values of raw pork and chicken were
0.50 and 0.61 and are in good agreement with our results.
There was a significant interaction (P < 0.01) between
method and storage time. The differences in TBARS
values of turkey raw meat from the two methods
increased as the storage time increased (Table 3). This
result indicated that even small changes in the status of
lipid oxidation in raw meat can be detected when
fluorometric method is used.
From the results, we concluded that the modified
fluorometric method can be successfully applied to
determine the lipid oxidation in raw meat. The method is
simple, reproducible, and sensitive, and requires small
sample volumes. Therefore, the modified fluorometric
method would facilitate the determination of oxidation
products in small quantities of samples. It is important,
however, to prepare both the reaction mixtures and
standards at the same pH.
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