6
ANALYTICAL BIOCHEMISTRY 206, 155-160 (1992) High-Performance Liquid Chromatographic Determination of N- -(2-Propenal)lysine in Biological Samples after Derivatization with Diethylethoxymethylenemalonatel Julio GirSn, Manuel Alaiz, and Eduardo Vioque Instituto de la Grasa y sus Derivados (CSIC), Apartado 1078, E-41012 Seville, Spain Received April 17, 1992 A recently reported methodology for amino acid anal- ysis by HPLC has been adapted for quantification of N-~-(2-propenal)lysine (a modified lysine by reaction with malondialdehyde that has been found in enzymatic digests of foods and in urine) in biological samples. We describe its use for investigating the in vitro degrada- tion of N-~-(2-propenal)lysine using rat tissue homoge- hates. Lysine dipeptide, used as a control in the incuba- tion mixtures, and the lysine released by the hydrolytic action of the homogenates in the in vitro incubations are quantified in the same way. The samples are sub- jected to a cleanup prederivatization step using PD-10 disposable columns (Pharmacia). This allows precol- umn derivatization with diethylethoxymethylenemal- onate (50 rain, 50°C) and resolution of the derivatives of the compounds of interest by reversed-phase HPLC (binary gradient, 45 min) with quantification based on the uv absorption of the derivatives at 280 nm (detec- tion limits below 1 pmol). The entire analysis takes 110 min. This method can be of general use for the determi- nation of N-~-(2-propenal)lysine in the context of re- search dealing with protein deterioration by reaction with malondialdehyde in biological systems and in foods. A method for the synthesis of N-~-(2-propenal)ly- sine, used as external standard for the HPLC analysis, is described. © 1992 Academic Press, Inc. Malondialdehyde (MDA) 2 is a toxic aldehyde gener- ated as a product of the oxidative decomposition of lipid peroxides in tissues and in foods and as a product of the cyclooxygenase reaction in prostaglandin metabolism ' This work has been supported by CICYT Grants ALI88-0169 and ALI91-0409. 2Abbreviations used: MDA, malondialdehyde; EPL, N-e-(2-pro- penal)lysine; DEEMM, diethylethoxymethylenemalonate; MDANa, malondialdehyde sodium salt; BHT, butylated hydroxytoluene. 0003-2697/92$5.00 Copyright@) 1992by Academic Press, Inc. All rights of reproduction in any form reserved. (1,2). Because of its high reactivity with functional groups of biological molecules, it has been investigated with regard to the biological and antinutritional effects that the reaction with free e-amino groups of proteins could have in vivo and in foods, respectively (3). Never- theless, little is known about the chemical structures that arise in these reactions. Several of these structures have been described as products isolated from chemical reactions between lysine esters, or related amino com- pounds such as butilamine, and MDA (4-6). These reac- tions are carried out as models of the reaction between proteins and this aldehyde (5,7-9). However, N-e-2-pro- penyl derivatives (10) are the only derivatives that have also been formed under real conditions. Draper's group found N-e-(2-propenal)lysine (ePL, see structure in Fig. 1) within the rat and human urinary metabolites of both in vivo and dietary proteins modified by MDA (11,12). In addition, ePL accounts for most of the reaction prod- ucts of MDA released by the in vitro enzymatic digestion of foods (13). ePL has been identified and determined in urine in the above-mentioned studies (11-13) using a procedure that includes anion-exchange and size exclusion chro- matographies and reversed-phase HPLC, with analysis of the collected fractions for MDA as the thiobarbituric acid derivative using HPLC. This procedure has been successful for the isolation and identification of ePL from urine. Nevertheless, it is too complex to be used as an anlytical method for routine ePL determination. In this paper we describe a simple and rapid proce- dure for the determination of ePL in tissue homoge- nates as used in the in vitro bioavailability investigation of this lysine derivative. Determination was achieved using a quick size exclusion chromatographic cleanup step, followed by derivatization with diethylethoxy- methylenemalonate (DEEMM) and quantification us- ing reversed-phase HPLC with uv detection at 280 nm. DEEMM is a universal reagent for amino groups and 155

High-performance liquid chromatographicdetermination of n-ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

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Page 1: High-performance liquid chromatographicdetermination of n-ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

ANALYTICAL BIOCHEMISTRY 206, 155-160 (1992)

High-Performance Liquid Chromatographic Determination of N- -(2-Propenal)lysine in Biological Samples after Derivatization with Diethylethoxymethylenemalonatel Ju l i o Gi rSn , M a n u e l Alaiz, a n d E d u a r d o V i o q u e Instituto de la Grasa y sus Derivados (CSIC), Apartado 1078, E-41012 Seville, Spain

Received April 17, 1992

A recent ly reported methodo logy for a m i n o acid anal- ys is by HPLC has been adapted for quant i f icat ion of N-~- (2-propenal ) lys ine (a modif ied l y s ine by react ion w i th malond ia ldehyde that has been found in e n z y m a t i c d igests of foods and in urine) in b io logica l samples . We descr ibe i ts use for inves t iga t ing the in v i t r o degrada- t ion of N-~- (2-propenal ) lys ine us ing rat t i s sue homoge- hates . L y s i n e dipeptide, used as a contro l in the incuba- t ion m i x t u r e s , and the lys ine re leased by the hydro ly t i c act ion of the h o m o g e n a t e s in the in v i t r o incubat ions are quantif ied in the s a m e way . The s a m p l e s are sub- jected to a c leanup preder iva t i za t ion s tep us ing P D - 1 0 disposable co lumns (Pharmacia) . This a l lows precol - umn der iva t i za t ion w i t h d i e t h y l e t h o x y m e t h y l e n e m a l - onate (50 rain, 50°C) and reso lut ion o f the d e r i v a t i v e s of the compounds of in teres t by reversed-phase HPLC (binary gradient , 4 5 min) w i th quanti f icat ion based on the uv absorpt ion of the der iva t ive s at 2 8 0 nm (detec- t ion l imi t s be low 1 pmol) . The ent ire ana lys i s t a k e s 110 min. This method can be of genera l use for the de termi - nat ion of N-~- (2-propenal ) lys ine in the con tex t o f re- search deal ing w i th prote in de ter iorat ion by react ion wi th ma lond ia ldehyde in bio logical s y s t e m s and in foods. A method for the synthes i s of N-~-(2-propenal ) ly- s ine , used as e x t e r n a l s tandard for the HPLC analys i s , is described. © 1 9 9 2 A c a d e m i c Press , Inc.

Malondia ldehyde (MDA) 2 is a toxic a ldehyde gener- ated as a p roduc t of the oxidative decomposi t ion of lipid peroxides in t issues and in foods and as a p roduc t of the cyclooxygenase react ion in pros tag landin metabol i sm

' This work has been supported by CICYT Grants ALI88-0169 and ALI91-0409.

2 Abbreviations used: MDA, malondialdehyde; EPL, N-e-(2-pro- penal)lysine; DEEMM, diethylethoxymethylenemalonate; MDANa, malondialdehyde sodium salt; BHT, butylated hydroxytoluene.

0003-2697/92 $5.00 Copyright @) 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

(1,2). Because of its high react ivi ty with funct ional groups of biological molecules, it has been invest igated with regard to the biological and an t inu t r i t iona l effects t ha t the react ion with free e-amino groups of p ro te ins could have in vivo and in foods, respect ively (3). Never- theless, little is known about the chemical s t ruc tures tha t arise in these react ions. Several of these s t ruc tures have been descr ibed as produc ts isolated f rom chemical react ions be tween lysine esters, or re la ted amino com- pounds such as but i lamine, and MDA (4-6). These reac- t ions are carr ied out as models of the react ion be tween pro te ins and this a ldehyde (5,7-9). However , N-e-2-pro- penyl derivat ives (10) are the only derivat ives t ha t have also been fo rmed unde r real condit ions. Draper ' s group found N-e-(2-propenal) lys ine (ePL, see s t ruc ture in Fig. 1) within the ra t and h u m a n ur ina ry metabol i tes of bo th in vivo and die tary pro te ins modified by MDA (11,12). In addit ion, ePL accounts for mos t of the react ion prod- ucts of MDA released by the in vitro enzymat ic digest ion of foods (13).

ePL has been identif ied and de te rmined in ur ine in the above-men t ioned studies (11-13) using a p rocedure tha t includes an ion-exchange and size exclusion chro- matographies and reversed-phase H P L C , with analysis of the collected f ract ions for MDA as the th iobarbi tur ic acid derivat ive using H P L C . This p rocedure has been successful for the isolat ion and ident if icat ion of ePL f rom urine. Never theless , it is too complex to be used as an anlyt ical m e th o d for rout ine ePL de te rmina t ion .

In this pape r we describe a simple and rapid proce- dure for the de te rmina t ion of ePL in t issue homoge- nates as used in the in vitro bioavailabil i ty invest igat ion of this lysine derivative. De te rmina t ion was achieved using a quick size exclusion ch romatograph ic c leanup step, followed by der ivat izat ion with d ie thyle thoxy- m e t h y l e n e m a l o n a t e ( D E E M M ) and quant i f ica t ion us- ing reversed-phase H P L C with uv detec t ion at 280 nm. D E E M M is a universal reagent for amino groups and

155

Page 2: High-performance liquid chromatographicdetermination of n-ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

156 GIRON, ALAIZ, AND VIOQUE

has been used in amino sugar (14) and amino acid (15) chemistry. We have reported the amino acid analysis of acid hydrolysates of proteins after derivatization with this reagent (16).

A method for synthesis of EPL used as external stan- dard in the HPLC quantification is also described. McGirr et al. (11) have obtained ePL by reaction of ly- sine and MDA sodium salt (MDANa) in sodium acetate buffer, pH 4.2, as described by Nair et al. (10), and pre- parative chromatography using anion-exchange col- umns and reversed-phase HPLC with water as eluent. We have developed a method in which only organic sol- vents are used. It diminishes difficulties due to ePL un- stability.

MATERIALS AND METHODS

Chemicals, instruments, and animals. DEEMM was obtained from Fluka (Bucks, Switzerland) and lysine dipeptide (~-N-L-lysyl-L-lysine dihydrochloride) from Sigma (St. Louis, MO). HPLC-grade acetonitrile was from Romil Chemicals (Loughborough, UK).

Disposable PD-10 columns (8 ml of Sephadex G-25) were purchased from Pharmacia (Upsala, Sweden). MN-kieselgel 60 (0.063-0.2 mm particle size) for col- umn chromatography and Alugram analytical plates (20 x 20 cm) with fluorescent indicator for TLC were ob- tained from Macherey Nagel (Duren, Germany).

A glass column (40 × 1.8 cm) for adsorption chroma- tography was from Afora (Barcelona, Spain). The HPLC system, with a 4-#m C18 column, and the data acquisition and processing facilities were previously de- scribed (16); solvents were filtered through 0.22-#m filters (Millipore, Bedford, MA) and degassed with he- lium. 1H and 13C NMR spectra were obtained on Varian XL-200 (Palo Alto, CA) and Bruker AC300 (Rheinstet- ten, Germany) spectrometers, respectively, using tetra- methylsilane as internal reference.

Rats were obtained from IFFA CREDO (Lyon, France).

Synthesis o[ ~PL. MDANa monohydrate (0.222 g, 1.5 mmol), prepared following the procedure of Kiku- gawa and Ido (17), and L-lysine HC1 (0.274 g, 1.5 mmol) were dissolved in methanol (75 ml) and shaken at 37°C for 24 h. The reaction was followed by measuring the characteristic uv absorption of MDANa (maximum at 267 nm) and N-2-propenals (maximum at 280 nm). The reaction mixture was concentrated under N2 and sub- jected to kieselgel 60 (45 g) column chromatography, eluting stepwise with 200 ml of chloroform:methanol (3:2, 1:1, 2:3, 1:2, and 1:3). The collected fractions were examined by TLC, developing with n-propanol:water (8:2), and spots were visualized by exposure to uv light at 254 nm or to thiobarbituric acid pulverization reagent (9). Some contained mixtures of compounds with R r 0.47 and 0.37 and were rechromatographied and examined in

the same way. TLC pure crystalline ePL was obtained after evaporating the solvent under N2 and drying under high vacuum those fractions that afforded a single spot of Rf 0.47.

Incubation media for in vitro metabolic experi- ments. The hydrolysis of EPL was studied in vitro us- ing rat homogenates prepared by the method of Finot et al. (18), with modifications. Male Wistar rats (200-250 g, 12-h fasted) were sacrificed by decapitation. Liver and kidneys were homogenized in 10 mM Na phosphate buffer, pH 7.4, containing 1 mM EDTA and 2 × 10 -2 mM butylated hydroxytoluene (BHT) to make a 20% (w/v) mixture. The intestinal mucosa homogenate was ob- tained from the scrapings of the small intestine of one rat homogenized in 10 ml of 200 mM Tris-HC1 buffer, pH 7.6, with 1 mM EDTA and 2 × 10 -2 mM BHT. A mechanically driven Teflon-glass homogenizer was used.

Standard incubation mixtures contained 0.29 ml of homogenate to which 850 nmol of EPL or lysine or 425 nmol of lysine dipeptide dissolved in 0.71 ml of homoge- nization buffer was added. Incubations were performed at 37°C for 30 min with continuous agitation and stopped by addition of 11 ml of 1 M sodium borate buffer, pH 9, containing 0.02% sodium azide.

Preparatory cleanup and derivatization of biological samples. DL-a-Aminobutyric acid (1700 nmol) ,used as internal standard for the HPLC analysis, was added to the buffer (11 ml) that was used to stop the incubations. Two milliliters of the resulting mixture was applied to a Pharmacia PD-10 disposable column with the same so- dium borate buffer as eluent. The first 6 ml was dis- carded, and the following 4 ml was derivatized.

The derivatization was carried out in the same buffer with 3.2 #1 of DEEMM (0.8 #l/ml) and shaking at 50°C for 50 min. Fifteen microliters of the resulting mixture was injected into the chromatograph.

Chromatography. HPLC was accomplished using a binary gradient of (A) 25 mM sodium acetate, pH 6, con- taining 0.02% sodium azide and (B) acetonitrile. Sol- vent was delivered at a flow rate of 0.9 ml/min as fol- lows: time 0.0-3.0 min, linear gradient from A:B (91:9) to A:B (86:14); 3.0-13.0 min, elution with A:B (86:14); 13.0-30.0 rain, linear gradient from A:B (86:14) to A:B (69:31); 30.0-50.0 min, elution with A:B (69:31). Detec- tion was by uv at 280 nm.

Preparation of calibration standards. Standard solu- tions of EPL and lysine dipeptide were subjected directly to derivatization and were chromatographed as de- scribed, with no pretreatment . Calibration curves were obtained plotting peak response

peak response

peak area

internal standard area × internal standard amount

Page 3: High-performance liquid chromatographicdetermination of n-ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

CHROMATOGRAPHIC DETERMINATION OF N-e-(2-PROPENAL)LYSINE 157

H R H ~C=N /

\C=C / ~H HO / ~H

imine

1,

H20

s r s I 3 _

H 3 4 / C H 2 - C H e - C H 2 - C H 2 - C H - C 0 0 H9 /C-N " 9 to

H~C-C "II 5e

0 ~ ' , 2 H I H6 enami Be . H8

: . . . . . . . . .

,h ' ' ' ' ; ' ' ' '

P P M

FIG. 1.

!

-0

1H NMR spectrum of ePL synthesized as described under Materials and Methods, obtained at 200 MHz in deuterated water.

versus s tandard amount , using DL-a-aminobutyr ic acid as internal s tandard.

RESULTS

Synthesis of ~PL. Chromatographica l ly pure ~PL was obtained by the reaction of M D A N a and lysine in me thano l and adsorpt ion chromatography , with a yield of 6%. Elementa l analysis was in agreement with the molecular formula of the hydrate. Calcd for C9H1402N 2 • 1.5 H20: C, 51.66; H, 8.19; N, 13.39. Found: C, 51.92; H, 7.79; N, 13.27. The 1H (Fig. 1) and 13C N M R (not shown) spectra suppor t this structure. As proposed by Nair et al. for enaminal - type adducts (10) and de- scribed by McGirr et al. for ePL and N-a-acetyl-e-(2- propenal) lysine (11), the 1H N M R spec t rum shows a trans-enamine-trans-imine t au tomer i sm in which the trans-enamine form is the preferred conformat ion . The tr iplets at 3.11 and 3.22 p p m and the doublets at 8.55 and 8.47 ppm are assigned to the enamine and the imine forms, respectively. 13C N M R chemical shifts (ppm, ob- ta ined in deutera ted water at 75.1 MHz) and assign- ments (see C number ing in Fig. 1) are as follows: 23.2, C7; 28.3, C8; 31.4, C6; 44.1, C5; 55.6, C9; 100.7, C2; 162.1, C3; 174.6, C10; and 192.3, C1.

Derivatization and HPLC analysis of ePL and lysine dipeptide standards. Standards of ePL and lysine di- peptide were derivatized by reaction with D E E M M and injected into the chromatograph . Bo th compounds af-

forded single peaks, which eluted at 17.7 and 44.8 min, respectively (Fig. 2). Different concen t ra t ions of these compounds and DL-a-aminobutyr ic acid, as in ternal s tandard, were derivatized in triplicate and ch romato - graphed. A m o u n t s in the range 1-1500 pmol were in-

£

0.33 1

0..30! I I

0.27 -

0.24 ~ 0

i I 10 20 30

T~IE (mn)

A

KK

I I 40 50

FIG. 2. HPLC elution pattern of a mixture of DEEMM derivatives of Tris (T), ePL (P), DL-~-aminobutyric acid (A), lysine (K), and lysine dipeptide (KK) standards. Each of these derivatives was ob- tained as described under Materials and Methods and shown to af- ford a single peak when chromatographed alone. Each peak repre- sents 530 (P, K), 265 (KK), 1062 (A), or 125,000 (T) pmol injected in a volume of 15 #l. This would be the amounts injected into the chro- matograph in the analysis of the incubation mixtures if the com- pounds were not degraded by tissue homogenates.

Page 4: High-performance liquid chromatographicdetermination of n-ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

158 GIRON, ALAIZ, AND VIOQUE

I

0.27

0.26

0.25

K

_d J2 10

. , , , . . , - - . i .

0.24~ 20 ..30 40 50

TIME (rnm)

FIG. 3. HPLC elution pattern of the incubation mixture blank corresponding to liver homogenate.

jected. External standard calibration curves were ob- tained from these runs. Linearity of the response was ascertained in this range (correlation coefficients >0.990) and response factors were 2.78 for ePL and 2.34 for lysine dipeptide. For lysine and DL-a-aminobutyric acid, calibration curves previously obtained in the same way were used; response factors were 1.86 and 1.00, re- spectively (16). The detection limits for the four com- pounds were below 1 pmol.

Cleanup, derivatization, and HPLC elution pattern of incubation media. Blank incubation mixtures pre- pared using liver, kidneys, and intestinal mucosa homog- enates were derivatized and analyzed by HPLC. Before this, a cleanup step was carried out by applying the sam- ples to Sephadex G-25 disposable columns, so that com- ponents of apparent M r higher than approximately 1000 were removed. The elution pat terns obtained show that no interfering peaks elute at, or close to, the retention times of ePL, lysine dipeptide, or DL-a-aminobutyric acid derivatives. There is no reagent peak on the chro- matograms, because DEEMM does not absorb at 280 nm (absorption maximum at 240 nm). Figure 3 shows one of the chromatograms obtained. A compound that elutes at 16.3 min in the chromatograms corresponding to intestinal mucosa homogenate (Figs. 2 and 4A) was identified as the derivative of Tris, which was used as a buffer in the preparation of this homogenate.

Determination of EPL, lysine, and lysine dipeptide in the in vitro incubations. The aim of this work was to develop a method for quantifying ePL and lysine in in- cubations of ePL with tissue homogenates. In addition, lysine dipeptide had to be incubated as a control of the incubation mixtures; under the conditions employed it must be 100% hydrolyzed by peptidases, releasing free lysine. Degradation of the two incubated compounds was assessed by measuring both released lysine and un- altered bound lysine.

The results of preliminary assays carried out to test the suitability of the method for the in vitro metabolic studies are presented in Tables 1 and 2 (see also Fig. 4). They show the accuracy of the analyses. As control measures, free lysine was incubated and determined in the same manner. Because the incubation mixtures contain endogenous lysine, the values afforded by blank incubation mixtures (no compound added) have been subtracted to obtain the concentrations of released (Ta- ble 1) or added (Table 2) lysine, ePL, lysine, and lysine dipeptide were also determined in substrate blanks (no homogenate added).

ePL derivative is stable in the derivatized incubation mixture samples for 24 h at room temperature. The other derivatives studied are stable for days at room temperature or weeks at 4°C.

DISCUSSION

The method described in this paper for preparation of EPL is simpler than the method previously reported

A

0.33

0.30

0.27

0.24- I I 0 40 50

B

Z

Ul

T

I I I 10 20 30

TIME (re,n)

I 0.28

0 10

K

A

I I

20 30 I l

4O 5O

TK~E (re,n)

FIG. 4. Elution pattern of two examples of the HPLC analyses carried out: incubation of {A) ePL with intestinal mucosa homogenate and (B) lysine dipeptide with kidney homogenate. Peaks are desig- nated as described in the legend to Fig. 2.

Page 5: High-performance liquid chromatographicdetermination of n-ε-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate

CHROMATOGRAPHIC DETERMINATION OF N-e-(2-PROPENAL)LYSINE 159

(11). The use of only organic so lvents for r eac t ion and for p r e p a r a t i v e co lumn c h r o m a t o g r a p h y has p r e v e n t e d p rob l ems due to lysine N - 2 - p r o p e n a l s u n s t a b i l i t y dur- ing sample process ing. In p r e l i m i n a r y assays us ing aqueous solvents , we obse rved in t e r conve r s ion be tween ePL and i ts i somer , N - a - ( 2 - p r o p e n a l ) l y s i n e , as de- scr ibed by D r a p e r ' s group (11,12), and decompos i t i on of these molecules r ende r ing low R r yel lowish compounds .

Der iva t i za t ion with D E E M M affords s tab le der iva- t ives t h a t have very good c h r o m a t o g r a p h i c behav io r in r eve r sed -phase H P L C and al lows uv de t ec t ion at 280 n m with no r eagen t peak on the c h r o m a t o g r a m s and with low de tec t ion l imits . By means of a c l eanup s tep us ing Sephadex G-25 d i sposab le co lumns before the de- r iva t iza t ion , c lean e lu t ion p a t t e r n s of i ncuba t i on mix- tu res have been achieved. Th is fact has a l lowed resolu- t ion of ePL, lys ine d ipept ide , and DL-a -aminobu ty r i c acid in i ncuba t ion mix tu re s p r e p a r e d us ing t i s sue ho- mogena tes , wi th no in te r fe r ing peaks . A l though the ePL der iva t ive decomposes fas te r t h a n the o the r de r iva t ives descr ibed in th i s p a p e r and those of amino acids (16) (s table for weeks a t room t e m p e r a t u r e ) , i ts s t ab i l i ty is enough for a u t o m a t i o n of the c h r o m a t o g r a p h i c proce- dure. We have used au toma t i c overn igh t s amp le injec- t ion into the H P L C column.

One p icomole of ePL (lower value in the range in which l inea r i ty has been proved) in jec ted in to the H P L C is equ iva len t to a concen t r a t i on in the or ig ina l sample of 1.6 n m o l / m l of incuba t ion mix ture . W e have checked t h a t the c o n c e n t r a t i o n of ePL needed to reach this a m o u n t for the in jec t ion may be lowered if the sam- ple is d i lu ted only four t imes before the PD-10 c l eanup s tep ( smal le r d i lu t ions are not su i tab le because of an excessive v iscos i ty for e lu t ing th rough PD-10 co lumns) . Thus , concen t r a t i ons as low as 0.6 n m o l / m l of ePL can be de tec ted in 1 ml of biological sample .

TABLE 1

Concentration of Released Lysine from ¢PL and from Lysine Dipeptide in the in Vitro Degradation Experiments

Incubated compound

Tissue homogenate ePL a KK b

Liver 4.5 ± 27.6 769.7 ± 38.2 Kidneys -14.9 ± 34.2 803.2 ± 27.1 Intestinal mucosa 3.3 ± 15.7 786.5 ± 62.1

Note. Values are expressed in nmol of released lysine (total - endog- enous lysine)/ml incubation mixture and represent the mean ± SD of incubation mixtures prepared in triplicate using tissue homogenates from one rat. Endogenous lysine was determined in blank incubation mixtures prepared using the corresponding tissues (181.5 ± 24.7, 346.7 ± 53.1, and 113.4 _+ 17.3 nmol/ml, mean ± SD, for liver, kidneys, and intestinal mucosa, respectively).

a 850 nmol/ml was incubated. b 425 nmol/ml was incubated.

TABLE 2

Concentration of ~PL, Lysine Dipeptide, and Lysine after the in Vitro Degradation Experiments

Incubated compound

Tissue homogenate ePL" KK b K a'c

Liver 732.3 ± 12.4 0 881.3 ± 36.6 Kidneys 787.2 ± 29.3 0 841.6 ± 25.5 Intestinal mucosa 756.1 ± 30.0 0 846.3 ± 20.7 Blank (without

homogenate) 843.0 ± 32.2 435.7 ± 21.1 865.7 ± 30.2

Note. Values are expressed in nmol of the incubated compound/ml incubation mixture and represent the mean ___ SD of incubation mix- tures prepared in triplicate using tissue homogenates from one rat.

a 850 nmol/ml was incubated. b 425 nmol/ml was incubated. c Concentrations of added lysine (total - endogenous lysine) are

given. Endogenous lysine was determined in blank incubation mix- tures prepared using the corresponding tissues (see values in Note to Table 1).

T h e e x p e r i m e n t s desc r ibed here were u n d e r t a k e n as p a r t of a p r o g r a m to eva lua t e the b ioava i l ab i l i t y of ePL (unpub l i shed resul t s ) in the con tex t of the s tudy of the d e t r i m e n t a l n u t r i t i o n a l effects of the r eac t ion of M D A with free lysine or e-amino groups of p r o t e i n s in foods. Cons ide r ing the c om pl e x i t y of the s a m p l e s in which ePL has been a n a l y z e d (d i lu ted whole t i s sue homogena t e s ) and i ts l imi t of de t ec t ion (d iscussed above), we t h i n k t h a t the m e t h o d can be app l i ed to o the r b iological sam- ples for the d e t e r m i n a t i o n of th is lys ine der iva t ive in bo th in vitro a n d in vivo b iochemica l s tudies . Some of these app l i ca t i ons could include the ana lys i s of ePL in biological fluids such as u r ine or b lood and of p r o t e i n enzymat i c h y d r o l y s a t e s [ePL is d e s t r o y e d by the usua l acid hydro lys i s for a m i n o acid ana lys i s (13)], as a m e a n of assess ing in vivo l ip id p e r o x i d a t i o n and the r e su l t ing p r o t e i n de t e r io ra t ion . T h e m e t h o d also could be app l i ed to d e t e r m i n i n g p r o t e i n a l t e r a t i on by r eac t ion wi th M D A in foods.

ACKNOWLEDGMENTS

The authors thank the Organic Chemistry Department and the Analytical Chemistry Department (Faculty of Chemistry, University of Seville) for 1H NMR spectrum and elemental analyses, respec- tively, and Dr. Francisco J. Hidalgo for obtaining 13C and ~H NMR spectra.

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