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Poult Sci 2008. 87:108-115. doi:10.3382/ps.2007-00158
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

Thiamin Phosphates in Egg Yolk Granules and Plasma of Regular and Embryonated Eggs of Hens and in Five- and Seven-Day-Old Embryos

S. Bäckermann, C. Poel and W. Ternes1

Center for Food Sciences, Institute for Food Toxicology and Chemical Analytics, University of Veterinary Medicine Hannover, Bischofsholer Damm 15/123, D-30173 Hannover, Germany

1 Corresponding author: waldemar.ternes{at}tiho-hannover.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The thiamin (TH), thiamin monophosphate (TMP), thiamin diphosphate (TDP), and thiamin triphosphate (TTP) content was analyzed in yolk plasma and yolk granules of unincubated and incubated chicken eggs and in 5- and 7-day-old chick embryos. Analytes were extracted from samples weighing approximately 1 g, with trichloroacetic acid (0.4 g/L), isolated with solid-phase extraction, and determined with HPLC. Before solid-phase extraction with a C-18 cartridge and a strong anion exchange cartridge, analytes were converted to thiochromes with cyanogen bromide. For HPLC, a C-8 pre-column and a C-8 column connected to a NH2 column were used with a fluorescence detector. No thiamin phosphates were found in egg yolk plasma or granules in native and embryonated eggs. The TH content in DM was 3,400 ± 700 ng/g for yolk plasma, 1,500 ± 140 ng/g for yolk granules of unincubated eggs, and 4,400 ± 1,100 ng/g for yolk plasma and 2,100 ± 450 ng/g for yolk granules of 5-d embryonated eggs. In chick embryos, TH as well as thiamin phosphates were found. The mean content in 5-d-old embryos was 1,200 ± 400 ng of TH and 8,000 ± 1,000 ng of TDP/g of DM. In 7-d-old embryos, the content of TH was 900 ± 200 ng, TMP was 400 ± 200 ng, and TDP was 5,500 ± 1,300 ng/g of DM. Thiamin triphosphate was not detected, and the minimum detectable limit for T was 0.3 ng/mL, for TMP 0.5 ng/mL, and for TDP 0.9 ng/mL, respectively. Recovery was 71% for TMP and 64% TDP in plasma and 58% TMP and 47% TDP in granules. It was demonstrated that only TH is present in egg yolk fractions in native and embryonated eggs of hens and that thiamin phosphates can be found in 5- and 7-d-old embryos, which indicates that they are synthesized by the chick embryo. According to its assumed unique role in highly specialized tissues, TTP was not found in early embryos.

Key Words: granule • plasma • embryo • chick • thiamin phosphate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In the body of various species, including man and fowl, thiamin can be found as nonphosphorylated thiamin (TH), thiamin monophosphate (TMP), thiamin di-phosphate (TDP), and thiamin triphosphate (TTP). In the organism, all forms can be converted by phosphorylation or dephosphorylation by specific enzymes. As far as it is known, TH and TMP appear to be biologically inactive (Ball, 2004). In contrast, TDP is the active coenzyme form and part of pyruvate dehydrogenase and {alpha}-ketoglutarate dehydrogenase, both enzymes of carbohydrate metabolism. In acetolactate synthetase, it takes part in the biosynthesis of Val and Leu (Lehninger et al., 1998). Less is known about the biological function of TTP. It is located in relatively high amounts in the nerve tissue and is suspected to play a role in stabilizing neural membrane potential and in the synthesis of neurotransmitters (Eichenbaum and Cooper, 1971; Bettendorff et al., 1990; Ball, 2004). According to the biological function of TDP and TTP, characteristic symptoms are seen in TH deficiency. It leads to disturbances in cell metabolism and histological damage to nerve tissues, which becomes apparent in typical clinical symptoms such as muscle weakness, gastrointestinal disturbances, neurological symptoms, appetite loss, and depression. Thiamin is therefore an essential vitamin for humans and animals and necessary in embryonic development and in adulthood (Bitsch, 1997).

So far, total TH content has been determined in whole eggs, egg albumen, and egg yolk, without regard to individual thiamin phosphate derivates (Elmadfa, 1985; Souci et al., 2000). The average total TH content in egg yolk is 2.71 to 2.9 µg/g of wet matter and in egg albumen 0.22 to 0.27 µg/g of wet matter (Hisil and Ötles, 1997; Souci et al., 2000). Therefore, egg yolk can be regarded as the major store site for TH. In the literature, no information about the TH content in the main yolk fractions, plasma, and granules or the presence of phosphorylated forms of TH in egg yolk can be found. Although the thiamin phosphates have been determined in several animal tissues and foodstuffs (Vanderslice and Huang, 1986; Miyoshi et al., 1990; Egi and Kawasaki, 2003), there is no information as to their content in chicken egg fractions. Because thiamin phosphates are essential for cell metabolism in the developing chick embryo, they must either be synthesized during development or be present in the freshly laid egg. To answer this question, we analyzed thiamin phosphates in several egg fractions. We focused on egg yolk, because it is the most important nutrient store site for TH and is utilized by the developing embryo during embryogenesis (Yoshizaki et al., 2004).

As in eggs, only the total TH content has been determined in various tissues of chicken embryos (Romanoff and Romanoff, 1967), but there was no information given as to the proportion of the different thiamin phosphates. Although Sanders (1980) and Sawano and Fujita (1981) concentrated on the localization and activity of thiamin pyrophosphatase, an enzyme which converts TDP to TMP, the only direct detection of TDP was in hearts of 20-d-old chick embryos (Olkowski and Classen, 1999). All other references which could be found dealt with TH and thiamin phosphate metabolism after birth or hatching in rodents and chickens but not in early stages of chicken embryogenesis.

The initial aim of this study was therefore to detect thiamin phosphates in plasma and granules of the egg yolk and embryonated egg yolk of hens to determine the ratio of nonphosphorylated forms to phosphorylated forms of TH and the ratio of these forms in granule and plasma fractions of egg yolk. The second objective was to determine thiamin phosphate contents in the early developing chick embryo to further observe whether thiamin phosphates are present in nutrition stores of the egg of the hen or are synthesized by the developing embryo. The answers to all these questions are lacking in literature, and thus this paper helps close some existing gaps.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Samples

All eggs were from barn fowl Gallus gallus. Unembryonated eggs (Lohmann Selected Leghorn, White Leghorn, KG-Nordmark-Landei GmbH & Co., Scheckendorf, Germany) were derived from caged hens. Daily TH intake of these hens was 0.49 mg, because they were fed 110 g of a standard layer feed containing 4.5 mg of TH/ kg of feed. The eggs were stored at +4°C in a refrigerator for no longer than 2 wk before analysis. Average total egg weight was 59.3 g, average egg yolk weight 14.7 g, and average weight of albumen 34.1 g.

Fertilized unembryonated eggs (specific pathogen-free, Lohmann Selected Leghorn White Leghorn, Lohmann Tierzucht, Cuxhaven, Germany) were taken from caged hens. Thiamin content in the standard layer feed of hens was 3.5 mg/kg of feed. Because they were fed 120 g, the daily TH intake was 0.42 mg. The eggs were incubated at the Poultry Clinic of the University of Veterinary Medicine Hanover for 5 or 7 d at a temperature of 37.5°C and a humidity of 50 to 60%. After the defined incubation period, they were analyzed immediately. Average total egg weight of 5-d embryonated eggs was 50.9 g, average egg yolk weight 11.5 g, and average weight of albumen 30.7 g.

Six plasma and 6 granule samples were analyzed from unembryonated and embryonated eggs. For measuring the TH content of embryos, 4 samples each having four 5-d-old embryos (4 x 4 = 16) and 2 samples each having two 7-d-old embryos and 2 samples each having four 7-d-old embryos (2 x 2 + 2 x 4 = 12) were analyzed. The average weight of 5-d-old embryos was 0.21 g and for 7-d-old embryos 0.93 g. The average DM, analyzed by the gravimetric method, was 6.03% for 5-d-old and 6.12% for 7-d-old embryos.

Extraction Procedure

The shells of unembryonated eggs were broken, and the albumen was poured into a beaker. The yolk was extracted by hand and washed thoroughly with deionized water. To remove adhering albumen and water, it was rolled on a paper towel. Next, with a spatula, the yolk membrane was pricked and the contents poured into another beaker. Afterwards, the yolk was mixed with the same volume of deionized water. Aliquots were put into 1.5-mL Eppendorf tubes and centrifuged (Zentrifuge 3200 Eppendorf, Eppendorf Vertrieb Germany GmbH, Wesseling-Berzdorf, Germany) with a relative centrifugal force of 8,000 for 10 min. The supernatant is called plasma, and the sediment is called granules. The plasma was collected with a pipette and put into a beaker, and the granules were washed with deionized water and again centrifuged. The supernatant was discarded. For analysis, approximately 1 g of plasma or granules was weighed in a glass centrifuge tube. Another 1 g of granule and 2 g of plasma samples were analyzed for their DM content with the gravimetric method (Matissek et al., 1992).

The eggshell of an embryonated egg was opened, and the embryo was taken out with tweezers. Adhering egg contents were thoroughly removed and washed off with deionized water. For analysis, 2 to 4 embryos were weighed together in a glass centrifuge tube. The sample was ground with an overhead stirrer, and about half of the sample was analyzed with the gravimetric method for DM content. The remaining aliquot was weighed into a glass centrifuge tube for analysis.

Egg yolk of embryonated eggs was separately poured into a beaker. Aliquots were centrifuged in 1.5-mL Eppendorf tubes in an Eppendorf centrifuge to separate plasma from granules as described earlier.

Four milliliters of ice-cold 0.4 g/L trichloroacetic acid (item no. 8789.2, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) was added to 1 g of plasma samples, 4 mL of ice-cold trichloroacetic acid to 1 g of granule samples, and 3 mL of ice-cold trichloroacetic acid to embryo samples. The sample was then ground for 30 s with an overhead stirrer (Ultra Turrax TP 18-10, IKA Labortechnik, Staufen, Germany). Residues on the stirrer were washed into the sample tube with ice-cold trichloroacetic acid. The tube was placed in a centrifuge (Hettich Universal 1200, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany) and centrifuged with a relative centrifugal force of 1,500 for 15 min. The supernatant was filtered through a glass funnel with a paper filter (type 595, 110 mm, Schleicher & Schuell, Whatman GmbH, Dassel, Germany) into a 10-mL volumetric flask, which was placed into a brown sample jar to protect the sample solution from light. The procedure was repeated once more with the sediment. The volumetric flask was then filled to volume with deionized water.

The whole extract was placed in a brown sample vial and, 0.5 mL of 0.3 g/L cyanogen bromide solution was added. The solution was adjusted to pH 10 with 1 M NaOH and left to stand for 20 min in the dark. After derivatization, the pH 10 was adjusted to pH 7 with 1 M hydrochloric acid.

For purification, a solid phase extraction (SPE) was performed with a strong anion exchange (SAX) cartridge (Discovery DSC-SAX, 3 mL, 500 mg, item no. 52664-4, Supelco, Sigma-Aldrich Chemie GmbH, Munich, Germany) and a C-18-endcapped cartridge (Chromabond, 3 mL, 500 mg, item no. 730013, Macherey-Nagel, GmbH & Co. KG, Düren, Germany) on a vacuum chamber (LiChrolut, Merck KGaA, Darmstadt, Gemany).

The cartridges were conditioned subsequently with 4 mL of methanol and 4 mL of deionized water. The C-18-endcapped cartridge was placed onto the SAX cartridge with a suitable connector. The cartridges were then placed on the vacuum chamber, and the sample was drawn through the cartridges. The analytes were then eluted with 4 mL of SPE-elution solution [20:80 (vol/ vol) acetonitrile:potassium phosphate buffer, 250 mM, pH 6.4] into a 5-mL volumetric flask and filled to volume with water.

Apparatus

Thiamin and thiamin phosphates were separated with a HPLC system and detected with a fluorescence detector (240 LC Perkin-Elmer, Perkin-Elmer LAS GmbH, Rodgau-Jügesheim, Germany). The HPLC system was composed of 2 HPLC pumps (model 64, Dr. Ing. H. Knauer GmbH, Berlin, Germany), a mixing chamber (Dr. Ing. H. Knauer GmbH), a C-8 precolumn (Eurospher 100, particle size 5 µm, 30 mm x 4.6 mm, item no. B120Y529, Dr. Ing. H. Knauer GmbH), and a C-8 column (Eurospher 100, particle size 5 µm, 150 mm x 4 mm, item no. B6Y529, Dr. Ing. H. Knauer GmbH), which was connected to a NH2 column. The NH2 columns from 2 different manufacturers were tested (first: NH2 LiChrospher 100, particle size 5 µm, 250 mm x 4 mm, item no. B748Y252, Dr. Ing. H. Knauer GmbH; second: NH2 Grom-Saphir 110, particle size 5 µm, 150 mm x 4 mm, endcapped item no. GSNHS0511S1504, Alltech-Grom GmbH, Rottenburg-Hailfingen, Germany). The whole system was operated with the Knauer Eurochrom 2000 software.

Chromatographic Conditions

If not stated otherwise, all solutions of substances were prepared in deionized water. Analysis was performed at a flow rate of 0.7 mL/min and a gradient over 45 min as can be seen in Table 1Go. Eluent A was prepared as 36:64 (vol/vol) acetonitrile (Fisher Scientific, Schwerte, Germany): 65 mM, pH 6.5 potassium phosphate buffer (potassium phosphate dehydrate, item no. 3904.1, Carl Roth GmbH & Co. KG; dipotassium phosphate hydrate: item no. 1.05104.1000, Merck KGaA). The final eluent was adjusted to pH 7.15 with 1 M HCl and 1 M NaOH. Eluent B was prepared as 40:60 (vol/vol) acetonitrile: 110 mM, pH 6.5 potassium phosphate buffer. The final eluent was adjusted to pH 7.25 with 1 M HCl and 1 M NaOH. The excitation wavelength was 375 nm and the emission wavelength was 450 nm.


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  		Table 1. High-performance liquid chromatography gradient table
 
After analysis, the columns were flushed with acetonitrile for 15 min and then kept overnight in a circular loop. Before resuming measurements, the columns were flushed with eluent until the base line was stable.

Calibration Solutions

Working calibration solution was prepared at a concentration of 1.1 µg/mL of TH (Merck KGaA), 2.8 µg/ mL of TMP (Sigma-Aldrich, Schnelldorf, Germany), and 4.6 µg/mL of TDP (BioChemica, Applichem GmbH, Darmstadt, Germany) by appropriate dilution of stock solutions containing 1.1 mg/mL of TH, 2.8 mg/mL of TMP, and 2.3 mg/mL of TDP. Stock solutions were prepared in advance in 5 mM hydrochloric acid solution (item no. 316.2500, Merck KGaA), divided into approximately 0.5-mL portions and stored frozen at –20°C. Each day, calibration standards were freshly prepared by thawing stock solutions to room temperature and diluting them as required.

From each of these working standard solutions, 400 µL was pipetted into a 5-mL volumetric flask. Two hundred microliters of 0.3 g/L cyanogen bromide solution (item no. 16774-25G, Sigma-Aldrich) and 200 µL of 1 M NaOH solution (item no. 6498.1000, Merck KGaA) were added, left for 20 min, and then eluent B was added to a final volume of 5 mL. Three more calibration solutions were prepared by diluting 1:1 with eluent B. A TTP (item no. 209-10841, Wako Chemicals GmbH, Neuss, Germany) solution of 1,000 mg/L was diluted to 600 ng/mL and independently analyzed to measure retention time.

Recovery Studies and Limit of Detection

For recovery evaluation, each of 16 plasma and 9 granule samples were spiked with 840 ng of TMP and 1,374 ng of TDP before extraction. The detection limit was calculated on the basis of regression lines for the standards and blank values (Gottwald, 2004).

Statistical Analysis

Data were analyzed for normal distribution with a Kolmogorov-Smirnoff test, for equality of variances with an F-test, and for significance of variation with the Student’s t test.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Egg Yolk Fractions of Unincubated and Incubated Eggs

Chromatograms of standard solutions (Figure 1Go) show an elution order of first TH, then TMP, followed by TDP. A simultaneous analysis of higher-phosphorylated forms of TH is still in development. The retention time of TTP, which was beyond 40 min, was determined apart from the other forms of TH. In chromatograms of plasma and granule fractions of regular and 5-d embryonated eggs, only 1 single peak, which is identified as TH, can be detected (Figures 2Go and 3Go).


Figure 1
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  		Figure 1. Chromatography of calibration solutions with different NH2-phase HPLC columns. T = thiamin (44 ng/mL); TMP = thiamin monophosphate (112 ng/mL); TDP = thiamin diphosphate (183 ng/mL).

 

Figure 2
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  		Figure 2. Thiamin in plasma and granules (1 g) of native egg yolk. T = thiamin.

 

Figure 3
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  		Figure 3. Thiamin in plasma and granules (1 g) of embryonated egg yolk. T = thiamin.

 
It is possible that thiamin phosphate contents in yolk fractions of regular and embryonated eggs were below the minimum detectable limit. Recovery was 71% for TMP and 64% for TDP in plasma and 58% for TMP and 47% for TDP in granules. The detection limit for TH was 0.3 ng/mL, for TMP 0.5 ng/mL, and for TDP 0.9 ng/mL, as was calculated on the basis of regression lines for the standards and blank values (Gottwald, 2004). This means that approximately 4 ng of TMP and 10 ng of TDP per gram of sample went unnoticed.

The TH content of plasma and granules in regular and embryonated eggs is summarized in Table 2Go. Mean DM was 22% for plasma and 45% for granules, which was analyzed with the gravimetric method (Matissek et al., 1992). Of course, these values differ from values that would be obtained if yolk is not diluted 1:1 to separate plasma and granules.


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  		Table 2. Thiamin content in yolk fractions of native and 5-d embryonated eggs
 
The ratio of TH in plasma and granule DM is approximately 2:1 in embryonated and unembryonated eggs. The mean content of TH in both plasma and granules is greater in embryonated eggs than in unembryonated eggs on a DM basis. The t-test results show no significant variation for TH in plasma, but for granules, the greater TH content is highly significant (P ≤ 0.01). As summarized by Naber (1993), the TH content in egg deviates by 64% and, according to Souci et al. (2000), ranges from 1.7 µg/g to 3.5 µg/g of wet matter. This deviation can, to some extent, be explained by differences in race and feed (Naber, 1993). Because external factor such as race and TH content in feed were the same for all laying hens, there must have been other unknown reasons for the greater TH content in embryonated egg yolk. However, it seems more likely that varying TH content in unincubated eggs, rather than changes in TH content during embryogenesis, was the reason for the different TH content.

On the basis of a 3:1 ratio of plasma to granules in egg yolk, it can be estimated that 87% of TH is stored in the plasma fraction and only 13% TH is stored in egg yolk granules. It therefore could be shown that egg yolk plasma is the storage site for TH. Further, it could be demonstrated that no TMP, TDP, or TTP could be detected in egg yolk fractions of unembryonated eggs and that no detectable quantities of thiamin phosphates are produced or stored during embryogenesis in the egg yolk fractions of embryonated eggs. Whether molt or age of hens have influence on total TH content in eggs would be of interest and could be the subject of further research projects.

Chicken Embryos

To improve separation of TH and thiamin phosphates, we tried another NH2 column from a different manufacturer. Retention time was confirmed each day with a calibration solution. As can be seen in Figure 1Go, retention time was slightly longer, but separation was better. It could also be observed that retention times decreased moderately after some months of column usage.

In contrast to the findings in yolk fractions of regular and embryonated eggs, thiamin phosphates were present in embryos. In 5-d-old embryos, a TH and a TDP peak were found, and in 7-d-old embryos, a small TH and TMP and a high TDP peak can be seen (Figure 4Go).


Figure 4
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  		Figure 4. Analysis of a sample (0.46 g) of 5-d-old embryos and a sample of 7-d-old embryos (1.75 g). T = thiamin; TMP = thiamin monophosphate; TDP = thiamin diphosphate.

 
It can be summarized that no phosphorylated forms of TH were found in yolk fractions and albumen (chromatograms not shown) in regular and embryonated eggs but in 5- and 7-d-old embryos. The synthesis of TMP and TDP by the developing embryo is the most likely explanation, because the egg is shut off from the outside environment by the eggshell.

As far as it is known, TMP plays no essential role for cell metabolism and accordingly could not be found in high amounts in the developing embryo. On the other hand, TDP is an essential coenzyme in energy metabolism and was therefore found in greater amounts. Thiamin triphosphate is found in adult chickens in high amounts in highly specialized tissues like muscles and nerve tissue. Miyoshi et al. (1990) found that 80% of the total TH content in white and 30% in red muscle is TTP. It is suspected to play a role in neural (Bettendorff et al., 1991) or membrane (Bettendorff et al., 1990) functions rather than energy metabolism. At this early stage of development, tissue differentiation has not proceeded, and therefore, the absence of TTP is not unexpected.

The amount of TH, TMP, and TDP found in embryos on the fifth and seventh day of incubation is shown in Table 3Go. The initial aim of this study was to answer the question of whether thiamin phosphates are present in regular eggs, embryonated eggs, or the developing embryo. In the nutrient storage site, namely the plasma and granules of yolk, thiamin phosphates were found neither in unembryonated nor embryonated eggs. In contrast, thiamin phosphates were detected in the developing embryo, and it is assumed that they were synthesized. As stated earlier, thiamin phosphates can be found in hearts of 20-d-old chick embryos (Olkowski and Classen, 1999) and in the muscles of adult chickens (Miyoshi et al., 1990). According to these articles, it was reasonable to believe that thiamin phosphates are formed during embryogenesis. But, there was no proof of this assumption found in literature, and it was unknown if thiamin phosphates are present in egg yolk, which could be used by the developing embryo. We filled this knowledge gap and showed that thiamin phosphates are not present in regular and embryonated egg yolk, that they can be detected in early embryos, and that it is reasonable to believe that they are synthesized by the developing embryo. The detection of thiamin phosphates can be related to the findings of Sanders (1980) and Sawano and Fujita (1981). The former proved the activity of the thiamin dephosphorylating enzyme thiamin pyrophosphatase in cultured chick embryo endoblast and mesoderm cells and the latter in chick embryo thyroid cells.


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  		Table 3. Thiamin and thiamin phosphate content in 5- and 7-d-old embryos1
 
Recovery

The recovery results were 70.5 ± 13% for TMP and 63.6 ± 16% for TDP in the plasma of egg yolk (n = 16) and 57.7 ± 15% for TMP and 46.8 ± 13% for TDP in granules of egg yolk (n = 9). We undertook great efforts in optimizing the recovery rates. In spite of this, the recovery of TMP and TDP in spiked samples was lower in previous reports in literature. In previous reports, recovery values are above 85% for urine (Losa et al., 2005), blood (Kimura and Itokawa, 1985; Bettendorff et al., 1986; Brunnekreeft et al., 1989; Tallaksen et al., 1991; Gerrits et al., 1997; Losa et al., 2005), organs (Batifoulier et al., 2005), beer and beer raw materials (Zapala, 2003), erythrocytes (Baines, 1985; Herve et al., 1994; Losa et al., 2005), nerve tissue (Bettendorff et al., 1991), and rat tissue (Sander et al., 1991). There are several possible explanations for the lower recovery results in our work. To evaluate decomposition of thiamin phosphates during extraction, we tested a low-concentration (8%) trichloroacetic acid solution and a high concentration (25%) and found that recovery in the latter was 13% lower for TMP and 20% lower for TDP. We therefore used a 4% trichloroacetic acid solution to prevent losses during extraction. To prevent degradation during extraction, samples were cooled, protected from light and alkaline conditions, and prepared immediately. With the same spiked solution analyzed before and after SPE, we could demonstrate that because recovery was above 85%, this was also not the reason for lost analytes. After all other possible reasons were excluded, we assumed that maybe the egg matrix itself was the problem. Vanderslice and Huang (1986) analyzed TH and thiamin phosphate contents in various foods. In their work, recovery was over 80%, for bread, cereal, almonds, and ham and pork loins but only 60% for chicken products. However, these results can not be fully compared with ours, because another extraction procedure was used. Because we analyzed other sample matrixes with the same method, recovery was above 90%, indicating that indeed differences in matrix composition result in lower recovery.


   ACKNOWLEDGMENTS
 
We wish to express our gratitude to the Poultry Clinic of the University of Veterinary Medicine Hanover for providing embryonated eggs. We also wish to thank the Fritz Ahrberg Foundation for the financial support.

Received for publication April 17, 2007. Accepted for publication September 27, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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