238

BBA 52793

Biochimrca et Biophysics Actu 959 (1988) 238-246

Elsevier

Fatty acids bound to ~-fetoprotein and albumin during rat development

Miguel Calvo a, Javier Naval ‘, Ferrnin Lampreave a, JosC Uriel b and And&s Piiiieiro a

o ~e~arta~e~to de Bjoqui~~c4 ~~iLle~s~dad de Zoragoza, Zaragoza ~S~~in~

and ’ Institut de Recherches Scientifiques SW le Cancer, Wejuif (France)

(Received 15 October 1987)

Key words: a-Fetoprotein; Albumin; Docosahexaenoic acid; Free fatty acid: Milk lipid; Development; (Rat)

The time-course levels and composition of the fatty acids bound to rat a-fetoprotein (AFP) and albumin from several sonrces, were determined throughout development, and related to the intake of lipids from milk and the com~sition~ changes in brain and liver fatty acids. The major fatty acids bound to AFP were polyunsaturated and mainly docosabexaenoic acid (22 : 6( n - 3)), either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22 : 6 (9.6%). Both AFP-eoncanavalin A microfo~s showed identical fatty acid com~sition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22: 6 by liver. Except for colostrum, levels of 22: 6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18 : 3 (n - 3)). These results support a specialized role of AFP in the plasma transport and tissue delivery of ~l~sa~at~ fatty acids, and mainly docosahexaenoic acid.

Introduction

a-Fetoprotein (AFP), a major glycoprotein of embryonic and fetal plasma, is distributed largely among all vertebrate species, which suggests an import~t role for this protein during develop- ment. However, the biological function of AFP is still incompletely elucidated.

It has been shown that AFP, like albumin, binds long-chain fatty acids, preferably long-chain

Abbreviation: AFP, cx-fetoprotein.

polyunsaturated fatty acids, namely, arachidonic (20 : 4( n - 6)) and docosahexaenoic (22 : 6( n - 3)) acids. The ability to bind fatty acids is, so far, the only property common to AFP from all species studied, including man [l], rat [2], mouse [3], calf [4], pig [5] and chicken [6]. Hence, this property could be related to the physiological role of AFP. The arachidonic and docosahexaenoic acids are derived from the essential fatty acids, linoleic (18 : 2(n - 6)) and linolenic acids (18 : 3(n - 3)) respectively, through sequential desaturation and elongation processes. In mammals, the arachidonic and docosahexaenoic acids are the main un- saturated acyl moieties of membrane phospholi- pids in cerebral cortex, retina, muscle, adrenals and testis 17-91. During development, the ability of the fetus to synthesize these fatty acids from

Correspondence (and present address): M. Calvo, Departa- mento de Tecnologia y Bi~ui~ca de 10s Alimentos, Facultad

de Veterinaria, Universidad de Zaragoza, Miguel Servet 171,

50013 Zaragoza, Spain.

0005-2760/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

239

their metabolic precursors is quite limited [lO,ll], which means that they must be incorporated by those tissues from external sources. Several auth- ors [11,12] have suggested that, at least in the rat, the preferred form of uptake of polyunsaturated fatty acids by the brain is as unesterified fatty acids. On the other hand, the intracellular pres- ence of AFP in fetal tissues of developing mammals, including brain and neural tube deriva- tives [13,14], has been well established. The occur- rence of AFP in developing brain cells seems due to protein uptake rather than local synthesis [15]. The transport of estrogens in rodents [16], or fatty acids in these and other species [2,5], has been repeatedly advanced as a possible explanation for the AFP uptake by the fetal cells.

In order to ascertain the possible role of AFP in the transport and transfer of polyenoic fatty acids to developing cells, it seemed worth analys- ing the steady-state of the fatty acid content of AFP and serum albumin from biological fluids (amniotic fluid and plasma) through rat develop- ment.

Materials and Methods

Animals and biological samples Inbred Wistar rats, fed with a standard labora-

tory chow (Pan-Lab, Barcelona, Spain) were used in all experiments. Pregnant rats (gestational age, 17-19 days) were anesthetized with sodium pentobarbital (30 mg/kg body wt., i.p.), and fetuses were delivered by cesarian section. Amniotic fluid was obtained by punction of the amniotic sac, and those with visible blood con- tamination were discarded. Blood samples were obtained from fetuses, neonates or adults by de- capitation under diethyl ether anesthesia, and sera were obtained by low-speed centrifugation after blood coagulation. Brain and liver were obtained from the same animals by dissection, washed with cold phosphate-buffered saline (PBS), blotted dry, and frozen. Samples of colostrum and milk were obtained from the stomach content of pups every 2 days from birth until the 18th day post-partum. The samples of serum, amniotic fluid and milk were also stored frozen at - 30 o C until their use.

a-Fetoprotein and albumin isolation Previous work from our laboratory [17] has

demonstrated that isolation of AFP (and albumin) by direct immunoadsorption and acidic elution results in a great loss of the bound fatty acids, and particularly of the polyunsaturated ones (up to 80%). Therefore, in the present study, we have purified AFP and albumin by a combination of several non-denaturing chromatographic steps, avoiding the exposure of proteins to high or low pH. A similar procedure has been used for the isolation of AFP and albumin from pig serum [S] and fetal rat extracts [17].

Pooled amniotic fluid (lo-20 ml) or sera (3-5 ml) were dialyzed against 25 mM potassium phos- phate/50 mM NaCl (pH 6.2) and then applied to a column (2 x 20 cm) of DEAE-Sephadex A-50 (Pharmacia, Sweden) equilibrated in the same buffer. The gel was washed with 100 ml of this buffer to eliminate unbound proteins. Bound pro- teins were eluted with a linear gradient of NaCl, from 50-400 mM in the same phosphate buffer (100 ml each). AFP and albumin were detected in the eluate by immunodiffusion using appropriate specific antisera. Fractions containing AFP and albumin were pooled and concentrated by ultra- filtration in cells equipped with PM-10 Diaflo membranes (Amicon, France). This mixture was dialyzed against 10 mM NaCl/SO mM Tris-HCl (pH 8.0) buffer and applied to a column (2.4 x 20 cm) of Cibacron Blue Sepharose, prepared as pre- viously described [18], and equilibrated in the same Tris buffer. Excluded fractions (3 ml each) containing AFP were pooled. In some cases, AFP was further purified to eliminate traces of protein contaminants by negative immunoadsorption on Sepharose-insolubilized antibodies against adult rat serum. Albumin bound to insolubilized Cibacron Blue was eluted with 0.2 M NaSCN/O.OS M Tris-HCl (pH 8.0) buffer. Fractions containing albumin were quickly dialyzed at 4 o C against 0.01 M potassium phosphate/O.15 M NaCl (pH 7.4) (phosphate-buffered saline) and concentrated by ultrafiltration. Albumin was finally purified by gel filtration in a column of Sephadex G-100 (2 x 70 cm) equilibrated in phosphate-buffered saline.

AFP and albumin were isolated also from a phosphate-buffered-saline homogenate of 17-19- day-old rat fetuses (2 : 1, w/v). The homogenate was centrifuged (18000 X g, 30 min, 4O C) and the supernatant dialyzed exhaustively against 25 mM

240

potassium phosphate/50 mM NaCl (pH 6.2)

buffer and subjected to the chromatographic treat-

ment described above. AFP and albumin obtained by this procedure

were 97-99% pure, as determined by polyacryl-

amide-agarose gel electrophoresis [ 191.

Lipid analysis

Total lipids were extracted, at 4” C, from rat milk, brain and liver by direct homogenization of

each gram of frozen sample in 20 ml of CHCl,/ CH,OH (2: 1, v/v) containing 10 mg of 2,6-di- tert-butyl-4-methylphenol per 1 of solvent, accord-

ing to the method of Folch et al. [20]. Fatty acids

bound to AFP and albumin (4-6 mg each) were extracted with isopropanol/n-heptane/ 0.5 M

H,SO, (4: 1: 1, v/v) as described previously in

detail [5]. In one experiment, the lipids bound to AFP isolated from a fetal homogenate were

extracted with chloroform/methanol (2 : 1, v/v) and analysed by thin-layer chromatography in silica-gel G plates (Merck, F.R.G.), using hexane/ diethyl ether/ acetic acid (70 : 30 : 1, v/v) as eluent.

Plates were stained with 0.1% of 2’,7’-dichloroflu- orescein in methanol and examined under ultra-

violet illumination. Visible spots were scraped out and extracted with chloroform.

In all cases, extracted lipids were transesterified

under nitrogen atmosphere at 80” C with H,SO, in anhydrous methanol for 2 h. The concentration

of sulfuric acid utilized was 0.5% (v/v) for lipids

from AFP and albumin, and 5% (v/v) for brain, liver and milk lipids. Fatty acid methyl esters were

analysed by gas chromatography in a column of 10% EGSS-X on Chromosorb W AW 100/200 and quantified by automatic integration using n-

heptadecanoic acid (17 : 0) as internal standard. Chromatographic peaks were identified by com- parison with the appropriate standards of known composition (Supelco, Bellefonte, U.S.A.). The gas chromatograph and the running parameters were the same as previously reported [5]. To determine the total amount of fatty acids bound per mol of protein, a molecular weight of 65000 for albumin

and 70000 for AFP were used.

Other methods Antisera against AFP, serum albumin and adult

rat serum were raised in rabbits as described pre-

viously [21]. Antibodies to rat serum were purified from the corresponding antiserum by the method of Avrameas and Ternynck [22] using insolubi-

lized adult rat serum as adsorbent, and later im- mobilized on Sepharose 4B by the CNBr method

[23]. Concanavalin A was isolated from jack bean meal (Sigma, U.K.) and insolubilized on CNBr- activated Sepharose 4B, as described elsewhere

[24]. The affinity derivative contained 12 mg of concanavalin A per ml of gel. AFP from pooled amniotic fluid was applied to a column of this derivative and eluted with 0.05 M Tris-HCl/l M NaCl/l mM CaCl,/l mM MnCl,/O.2% chlore-

tone (pH 7.5) buffer. Two AFP microforms, dif-

fering in their glycan moiety, were separed by this procedure: AFP not retained by concanavalin A

(65% of total), and AFP bound to concanavalin A (35% of total). Bound AFP was desorbed with 0.1

M methyl a-D-glucopyranoside (Sigma) in the same Tris-HCl buffer. Purified proteins were quantified by electroimmunodiffusion [25] using appropriate standards, previously quantified by the method of Lowry et al.

Results

Fatty acids bound to AFP and albumin isolated from amniotic fluid, fetal serum or whole fetuses

Thin-layer chromatography analysis confirmed that the only class of lipids associated with rat

AFP was the free fatty acids. Table I shows the composition of the fatty acids bound to AFP and albumin isolated from several sources: fetal homo- genates, fetal serum and amniotic fluid. Serum AFP contained around 1.4 mol fatty acid per mol protein, of which 37% were polyunsaturated. AFP from whole fetuses exhibited the maximum con- tent of polyunsaturated fatty acids of chain-length 20 carbon atoms or longer (46.3% of total fatty acids). The major fatty acid bound to AFP, either from serum or fetal extract, was docosahexaenoic acid (22 : 6( n - 3)), which represented approx. 23% and 22%, respectively, of total fatty acids. The major fatty acids bound to albumin, from the same samples, were palmitic (16 : 0) and oleic (18 : l(n - 9)) acids. Except in the case of albumin from fetal extract, this protein carried very few polyunsaturated fatty acids; among these, the only one found in a significant amount was arachidonic

241

TABLE I

FATTY ACIDS BOUND TO a-FETOPROTEIN AND ALBUMIN FROM AMNIOTIC FLUID, FETAL SERUM AND FETAL

EXTRACT

Results are expressed as percent (by weight) of total fatty acid content and are the mean values of the analysis of two (fetal serum

and fetal extract) or four (amniotic fluid) independent pools. S.D. was less than 10% of the mean for components greater than 8% of

total fatty acids and less than 15% for minor components. The total value determined using 17 : 0 as internal standard is expressed in

mol of fatty acid per mol of protein. PUFA, polyunsaturated fatty acid. n.d., not detected.

Fatty acid a-Fetoprotein

amniotic

fluid

fetal

serum

fetal

extract

Albumin

amniotic

fluid

fetal

serum

fetal

extract

16:0 28.9 20.3 16.2 35.4 34.1 13.5

16:l(n-7) 10.8

18:0 4.8

lS:l(n-9) 20.5

18:2(n-6) 7.2

18:3(n -3) n.d.

20:4(n -6) 8.4

22:4(n -6) 3.6

22:5(n-6) 2.4

22:6(n-3) 9.6

Others 3.6

5.8 7.3 11.1

6.2 5.6 4.0

18.7 12.1 29.9

7.4 8.4 12.8

n.d. 0.3 n.d.

4.6 10.4 4.6

4.7 5.1 n.d.

4.5 5.5 n.d.

23.1 21.6 nd.

2.8 3.4 1.3

7.8 4.6

8.6 4.6

33.1 29.9

8.4 15.0

n.d. 0.5

3.8 15.4

n.d. 1.0

n.d. 1.7

n.d. 2.9

4.2 4.2

‘K, of PUFA 24.0 36.9 46.3 4.6 3.8 22.9

Total 0.8 1.4 1.7 2.3 2.7 2.8

acid (20 : 4(n - 6)). On the other hand, AFP from amniotic fluid contained only 0.8 mol of fatty acid per mol of protein. The major differences in fatty acid composition of AFP from fetal serum and

amniotic fluid occurred in the polyunsaturated fraction, and mainly in the amount of 22 : 6 bound.

While, in serum AFP, this fatty acid accounted for 23% of total fatty acids, this value dropped to 9.6% in amniotic fluid AFP.

Since AFP from serum and amniotic fluid are glycosylated differently [26], we investigated fur-

ther to see whether this difference could modify the fatty acid binding ability of AFP. Conse- quently, purified AFP from amniotic fluid was separated into two microforms, differing in their glycan structure [27], by affinity chromatography on insolubilized concanavahn A. Gas-chromatog- raphy analysis revealed the same fatty acid com- position, either qualitative or quantitative, for both AFP microforms.

Fatty acids bound to serum AFP and albumin through postnatal life

Tables II and III show the time-course com-

position of the fatty acids bound to AFP and

albumin from rat serum at different postnatal ages. Even though the major fatty acids trans-

ported by both proteins in this period were 16 : 0, 18 : 1 and 18 : 2, which were also the major acids of milk ingested by pups (Table IV), AFP from

newborn serum was also rich in 22 : 6. The com- position of fatty acids bound to this protein (Ta- ble II) varied through postnatal development and, particularly, with the percentage of polyun- saturated fatty acids. The maximum content of these fatty acids in serum AFP was found in the fetal period, decreasing thereafter until reaching a minimum percentage (3.1%) at the 8th day post- partum, and then increasing again. These changes corresponded mainly with the variation in levels of 22: 6, which represented 23% of total fatty

acids in AFP from fetal serum, 10.5% in newborn serum, and decreased to 0.6% in AFP from serum of 8-day-old rats. Conversely, the proportion of the rest of fatty acids increased, compensating for the relative diminution of 22 : 6. The total amount of fatty acids bound to serum AFP was about 1.6 mol per mol of protein, remaining with small

242

TABLE II

FATTY ACIDS BOUND TO RAT SERUM AFP THROUGHOUT THE POSTNATAL DEVELOPMENT

Data for the individual fatty acids are expressed as percent (by weight) of the total fatty acids. The total value is expressed in mol of

fatty acid per mol of protein. The results are the mean of three individual determinations of two independent pools. SD. was less

than 12% of the mean for components greater than 6% of total fatty acids and less than 15% for minor components. nd., not

detected. PUFA, polyunsaturated fatty acids.

Fatty acid

16:0

16:l(n-7)

18:0

18:l(n -9)

18:2(n -6)

20:4(n -6)

22:4(n -6)

22:5(n -6)

22:6(n -3)

Others

% of PUFA

Total

Serum AFP (wt%)

Age (days): 0 4 8 12 16 20

33.5 37.7 32.7 41.1 33.1 40.1

5.9 4.6 11.0 8.1 8.5 4.8

7.5 5.4 8.6 10.4 9.3 11.2

14.7 22.7 26.5 19.9 21.9 15.4

14.5 9.1 15.0 10.0 15.4 14.0

1.8 3.2 0.9 2.8 2.3 3.2

6.3 2.3 1.6 2.6 1.3 3.1

2.5 nd. n.d. 1.2 1.2 n.d.

10.5 2.9 0.6 1.3 6.1 5.5

2.9 2.1 3.1 2.6 1.8 2.7

21.1 8.4 3.1 7.9 10.9 11.8

1.7 1.6 1.5 1.8 1.3 1.7

variations throughout the postnatal period. The lated from amniotic fluid (Table I) or from serum amount of polyunsaturated fatty acids in serum (Table III) were also negligible in comparison with albumin was negligible, except for the arachidonic that of AFP. Only in adult rat serum, in which the acid. The levels of 22: 6 bound to albumin iso- levels of AFP are very low (less than 50 ng/ml),

TABLE III

FATTY ACIDS BOUND TO RAT SERUM ALBUMIN THROUGHOUT POSTNATAL DEVELOPMENT

Data for the individual fatty acids are expressed as percent (by weight) of total fatty acids. The total value is expressed in mol fatty

acid per mol of protein. Results are the mean of three individual determinations of two independent pools. S.D. was less than 10% of

the mean for components greater than 6% of total fatty acids and less than 15% for minor components. n.d., not detected. PUFA,

polyunsaturated fatty acids.

Fatty acid Serum albumin (wt%)

16:0

16:l(n -7)

18:O

lS:l(n -9)

18:2(n -6)

18:3(n -3)

20:4(n -6)

22:4(n -6)

22:5(n -6)

22:6(n -3)

Others

Age (days): 0 4 8 12 16 20 adult pregnant

32.4 30.6 52.5 37.0 36.5 20.0 22.1 32.1

16.3 5.9 1.4 3.4 5.1 1.8 0.7 2.3

4.4 9.8 7.0 7.4 10.0 15.3 16.5 12.4

14.8 25.1 18.8 25.4 23.2 31.9 29.4 26.1

23.3 18.7 13.8 19.3 17.5 16.2 16.6 15.1

0.8 1.4 0.6 0.3 n.d. 1.2 1.4 n.d.

3.6 4.3 2.9 3.0 4.5 8.7 6.8 7.8

1.0 1.1 0.6 0.9 n.d. 1.6 1.2 n.d.

n.d. 0.6 n.d. 0.4 nd. n.d. n.d. n.d.

0.5 0.8 0.2 0.3 nd. 0.9 1.8 2.7

2.9 1.7 2.2 2.6 3.2 2.4 3.5 1.5

% of PUFA 5.1 6.8 3.7 4.6 4.5 10.3 9.8 10.5

Total 3.3 2.4 2.3 3.1 2.9 3.2 2.1 2.2

243

TABLE IV

COMPOSITION OF FATTY ACIDS THROUGHOUT LACTATION

Fatty acid or chain-length 16 carbon atoms or longer were extracted from total lipids of rat milk (stomach content of pups)

throughout lactaction. Values are expressed as percent (by weight) of total fatty acids and represent the mean of those obtained for

three different litters. * S.D. was less than 10% of the mean for components greater than 15% of total fatty acids and less thant 20%

for minor components. nd. not detected. PUFA, polyunsaturated fatty acids.

Fatty acid Composition (wt%)

Days after birth: 0 2 4 6 8 10 14 18

33.5 39.1 40.3 45.1 42.8 52.2 46.1 23.5

7.3

2.2

29.8

17.5

0.1

1.0

1.1

9.1

0.6

3.4

0.3

1.0

1.8

1.3

5.1 1.8 2.8 1.5 5.1

3.2 0.3 3.2 2.8 0.6

21.7 23.8 28.7 20.9 25.1

20.7 24.0 16.9 20.0 18.9

0.6 0.5 0.7 0.5 0.7

0.8 0.8 0.5 0.3 0.8

1.0 0.6 0.7 0.3 0.7

3.6 4.0 2.5 3.2 1.6

0.5 0.5 0.3 0.3 0.3

0.8 1.3 0.7 1.0 nd.

0.2 0.3 n.d. 0.2 0.4

0.5 0.8 0.3 0.5 0.5

1.2 1.1 0.9 1.3 0.8

0.6 1.1 1.5 1.5 1.7

1.1 3.7

4.9 7.4

16.6 18.7

19.9 17.4

0.7 0.7

n.d. * 1.3

0.3 0.5

1.2 1.6

0.5 0.3

n.d. 0.2

0.2 0.1

0.2 0.2

0.9 0.6

1.3 1.2

3.3 3.5

16:0

16:l(n -7)

18:0

18:l(n -9)

18:2(n -6)

18:3(n-3)

20:o

20:3(n -6)

20:4(n -6)

20:5(n -3)

22:4(n -6)

22:5(n -6)

22:5(n -3)

22:6(n -3)

Other

% of PUFA 17.3 7.8 8.6 5.4 6.8 4.3

was the amount of 22 : 6 bound to albumin slightly significant (around 1.8% of the total fatty acids for normal rats and 2.7% for pregnant rats).

Fatty acid composition of milk lipids Table IV shows the time-course composition of

fatty acids with a chain-length longer than 16

carbon atoms, extracted from milk lipids throughout rat lactation. Palmitic (16: 0), oleic

(18 : 1) and linoleic (18 : 2) acids were the major fatty acids in all cases. Colostrum was richer in polyunsaturated fatty acids (17.3% of total fatty acids) than milk (range: 3.357.88). Among the polyunsaturated fatty acids, arachidonic acid was the main component, representing around 9% of the total fatty acids in colostrum. The proportion of docosahexaenoic acid, 22 : 6, was about 2% in the first colostrum and 1% or less in milk. This amount was always, nevertheless, much greater than the corresponding value for its biosynthetic precursor, linolenic acid (18 : 3).

Fatty acid composition of brain and liver lipids The fatty acid composition of total lipids ex-

tracted from the liver and brain of newborn rats

(from birth to 20th postnatal day) showed (Table V), in general, a pattern similar to that described

: 0.6 - -14 z?

12 16 20

DAYS AFTER BIRTH

Fig. 1. Time-course changes in the accumulation rate of doco-

sahexaenoic acid (22 : 6( n - 3)) by brain (0) and liver (A) of

newborn rats, as compared with the relative levels of 22: 6

bound to serum AFP (0). Values for brain and liver corre-

spond with the daily increase of 22 : 6 in each organ, expressed

as the total weight gain in this fatty acid (in mg) per organ per

day. Data are mean of 4-6 organs at each time point. Values

for 22 : 6 in AFP are the same as those presented in Table II.

244

TABLE V

POLYUNSATURATED FATTY ACID COMPOSITION OF TOTAL LIPIDS FROM BRAIN AND LIVER THROUGH

POSTNATAL DEVELOPMENT OF THE RAT

Results are the mean of 4-6 independent analysis of individual organs. S.D. represented less than 12% of the mean for components

greater than 1 mg per g of tissue, and less than 20% for minor components. Data are expresed in mg fatty acid per g tissue. Only the

major polyunsaturated fatty acids, longer than 20 carbon atoms, are presented.

Fatty acid Composition (mg/g tissue)

Time after birth (days): 0 2 4 6 8 10 12 14 18 27

Brain” 20:4(n -6) 1.6 1.8 2.0 2.1 2.4 2.6 2.8 2.9 3.2 3.6 22:4(n -6) 0.6 0.5 0.5 0.5 0.6 0.6 0.6 0.7 0.8 1.1 22:5(n -6) 0.6 0.5 0.4 0.4 0.4 0.4 0.2 0.5 0.4 0.6 22:6(n -3) 1.6 1.7 1.8 1.9 2.3 2.6 3.2 3.2 3.3 3.6

Total 11.6 14.0 15.8 16.0 17.5 18.3 19.2 20.5 24.1 30.3

Liver 20:4(n -6) 2.6 7.4 6.7 6.3 4.7 4.5 6.1 5.9 5.0 4.0 22:4(n -6) 0.8 1.6 1.1 1.6 0.5 0.4 0.2 0.4 0.3 0.4 22:5(n -6) 0.5 1.0 0.9 1.0 0.3 0.4 0.2 0.4 0.9 0.6 22:5(n -3) 0.2 1.1 0.9 1.3 0.5 0.4 0.4 0.6 0.8 0.4 22:6(n -3) 1.1 5.2 4.5 5.0 4.1 3.2 4.0 4.1 2.8 2.0

Total 21.1 47.3 38.4 43.4 28.0 24.8 29.5 30.0 29.2 23.0

a The amount of 22 : 5( n - 3) in brain was always less than 70 pg/g of tissue, in the period studied

in other previous works [2,7,28]. In the brain, the relative content of fatty acids, and mainly that of polyunsaturated fatty acids, was augmented through postnatal development. In particular, the amount of docosahexaenoic acid (22 : 6) demon- strated a strong accretion in this period, with a maximal increase rate between days 8 and 12 post-partum (Fig. 1). Considering the increase in brain weight in these 4 days, the amount of 22 : 6 in brain duplicates. In liver, there was an abrupt increase of its fatty acid content, and in particular, in the amount of 22: 6, from birth to the 2nd postnatal day. Afterwards, even though the 22 : 6 concentration remains almost constant, the accu- mulation rate of 22 : 6 by liver diminishes until the 12th day post-partum (Fig. l), in which a new transient increase of 22 : 6 accumulation occurred.

Discussion

In most vertebrate species, the AFP gene is expressed mainly in the fetal period. However, in species such as rodents, newborns are relatively immature, and AFP is actively synthesized also during the early postnatal period. This offers a suitable model to study the relationship between

the AFP metabolism and its putative biological effects. Furthermore, in recent years, evidence has been gathered supporting the view that arachidonic and docosahexaenoic acids, which are derived in adults from linoleic and linolenic acids, respec- tively, are also essential themselves for fetal devel- opment [28-301. During the lactation period, the sole source of essential fatty acids for newborns is the mother’s milk. In this period, there is a strong accumulation of polyunsaturated fatty acids in some rat tissues, such as the brain [2,31]. The specific activity for desaturation of 18 : 2 and 18 : 3 by homogenates of brain increases from birth until a maximum at 4 days postnatal, and de- creases sharply thereafter, coinciding in time with a rise in the specific desaturation rate by liver [lo]. However, the total desaturation capacity of brain remains low and almost constant over the 4-20- day period [lO,ll]. Total liver activity is similar to that of brain until 8 days of age, when it increases rapidly [lO,ll]. On the other hand, the incorpora- tion of 22 : 6 in brain is 60-times greater when administered in a preformed form than when an equivalent dose of linolenic acid is included in the diet [32].

According to previous data [l-5], the results

245

obtained here demonstrate that, in developing

mammals, the plasma transport of 22 : 6 is per- formed mainly by AFP, in spite of the higher levels of albumin. It is worth noting that the fatty acid content of AFP from amniotic fluid was only about half of that of AFP purified from the corre-

sponding fetal serum or whole fetuses. This loss of fatty acids was more marked for the polyun-

saturated fatty acids of the n - 3 series, and espe- cially for 22 : 6, which represented only 30% of its

corresponding value in serum. These differences

cannot be related to a distinct fatty acid binding ability exhibited by AFP microforms, since both AFP microforms, separated by concanavalin A,

showed identical fatty acid composition. This agrees with previous observations of other authors, showing that different concanavalin A microforms

of rat AFP exhibited the same affinity for estro- gens [33,34] which compete with fatty acids for the same binding site of AFP in rodents [35].

AFP in amniotic fluid can arise from fetal urine [36], gastrointestinal tract [37], skin transudation

[38] and, in rodents, probably also by direct diffu- sion from the yolk sac [39]. In this species, and contrary to in man and primates, this organ

synthesizes large amounts of AFP during the en- tire gestation [37], and seems to be the major contributor to amniotic fluid AFP. Therefore, the low content of 22 : 6 observed in this protein may be due to its direct transfer through yolk sac membrane before AFP can incorporate this fatty

acid from the surroundings or specialized tissues, such as liver or placenta. Alternatively, AFP could

interact with different fetal tissues, which would take up fatty acids from AFP, to be wasted after- wards in amniotic fluid, which could be consid-

ered, in this way, as a residual pool for proteins. We have studied in detail the time-course com-

position of fatty acids bound to serum AFP and albumin throughout rat development in order to correlate its changes with other metabolic

processes. The results obtained suggest a meaning- ful metabolic model of regulation of fatty acid transport and delivery to tissues. The total amount of fatty acids bound to AFP seems to remain relatively constant. However, the relative amount of polyunsaturated fatty acids, and especially 22 : 6, decreases from the fetal period to birth. It is known that the mother can transfer free fatty

acids (and the unsaturated ones easily) to the fetus

through the placenta [40]. The decrease in polyun- saturated fatty acids content of AFP from new- born serum could be explained by the change in the supply source of fatty acids, being in the fetus

the placental transfer, and the lipolysis of milk triacylglycerols in neonates. Throughout the post- natal period, the proportion of 22: 6 bound to

AFP decreases until reaching the lowest value,

actually trace amounts, at the age of 8 days. This diminution of 22 : 6 levels has been also observed

in total serum by other authors [41]. During this period, the serum AFP concentration remains

elevated and near constant at around 3 mg/ml [42]. On the other hand, our data indicate (Table

IV) that the concentrations of 22 : 6 and of any of

its possible precursors in rat milk are fairly con- stant through the suckling period. This fact has

also been observed in a recent study on the fatty acid composition of milk from rats fed with a

different standard chow [43]. Therefore, the changes in levels of the 22 : 6 bound to AFP must be due to (a) differences in the rate of incorpora- tion of this fatty acid to the tissues during post-

natal development of the rat or (b) differences in

the rate of synthesis and/or release of 22 : 6 from adipose tissue and liver, which could act as a

temporary adipose tissue in this period because, contrary to the adult status, a great part of hepatic 22: 6 is associated with triacylglycerols [7]. The

brain can incorporate the fatty acids found in blood as unesterified fatty acids [11,12]. These are

insoluble at the blood pH and they must be car- ried bound to proteins. The fatty acid composition

of the carrier proteins (AFP, albumin), shown in Tables II and III, may be considered representa-

tive of the steady state of the metabolic turnover of these lipids. Among the plasma proteins that can carry unesterified fatty acids, only AFP binds a significant amount of 22 : 6. The maximum rate of accumulation of this fatty acid by the brain occurs around 8-10 days post-partum. This maxi- mum is coincident in time with the minimal

amount of this fatty acid bound to serum AFP, and with a loss of 22 : 6 from hepatic stores (Fig.

I)- All these results, taken together, agree and sup-

port the hypothesis advanced in previous works [2,5,14] about the role of AFP as a specialized

246

carrier of polyunsaturated fatty acids, and mainly of 22 : 6, in developing tissues.

Acknowledgements

This work was supported in part by a grant

from the Fondo de Investigaciones Sanitarias de

la Seguridad Social (Health Ministry, Spain) and a Programme Commun de Recherche B Caract&-e

Prioritaire France-Espagnol.

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