ethanol intake during lactation alters milk nutrient ... · azara et al. biol res 41, 2008, 317-330...

317AZARA ET AL. Biol Res 41, 2008, 317-330Biol Res 41: 317-330, 2008 BREthanol Intake during Lactation Alters Milk NutrientComposition and Growth and Mineral Status of Rat Pups

CÍNTIA R.P. AZARA1, INGRID C. MAIA1, CAROLINA N. RANGEL1,MÁRIO A.C. SILVA-NETO2, RENATA F.B.SERPA3, EDGAR F.O. DE JESUS3,MARIA G. TAVARES DO CARMO1 and ELIANE FIALHO1*

1 Instituto de Nutrição Josué de Castro, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro,RJ, Brazil2 Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Centro de Ciências daSaúde, Universidade Federal do Rio de Janeiro, RJ, Brazil3 Laboratório de Instrumentação Nuclear / COPPE, Universidade Federal do Rio de Janeiro, RJ, Brazil

ABSTRACT

Lactating Wistar rats were fed a liquid diet containing either ethanol [ethanol-fed group (EFG)] or anisocaloric amount of carbohydrate [pair-fed group (PFG)] from day 1 postpartum up to day 14 of lactation, toinvestigate micro/macronutrient milk composition and the mineral status of pups. EFG presented a reductionof daily milk production and milk composition was significantly higher in protein and lower in carbohydrate,while the lipid content was similar to that of PFG. When compared to PFG, the milk of EFG had a decreasedproportion of C22:6 n-3 fatty acid and an increase in medium-chain fatty acids and of several minerals.Pups of EFG showed reduced growth and a lower concentration of Cu and Sr in plasma and lowerconcentrations of Ca, P and Cl, and higher concentrations of Cd in the brain. We conclude that maternal EtOHintake greatly impairs lactational performance and modifies the mineral status of pups.

Key terms: breast milk, ethanol, rats, lactation, minerals status and milk composition.

* Corresponding author: Departamento de Nutrição Básica e Experimental, Instituto de Nutrição Josué de Castro, Centrode Ciências da Saúde, Universidade Federal do Rio de Janeiro, UFRJ, Caixa Postal 68041, Cidade Universitária, Ilha doFundão, Rio de Janeiro, CEP 21941-590, Brasil. Telephone number + 55 21 2562 6449. Fax number + 55 21 2562 6695.E-mail address: [email protected]

Revised: August 8, 2008. In Revised form: August 22, 2008. Accepted: September 2, 2008

ABBREVIATIONS: AA - arachidonic acid; AIN-93G - American Institute of Nutrition; DHA -docosahexaenoic acid; EFG - ethanol-fed group; EPA - eicosapentaenoic acid; eV - electron-volt; Ga - gallium;IP - intraperitoneal; keV - kilo electron-volt; mrad - mili roentgen absorbed dose; MUFA - monounsaturatedfatty acid; PFG - pair-fed group; PUFA - polyunsaturated fatty acid; SFA - saturated fatty acid; TXRF - TotalReflection X-Ray Fluorescence.

1. INTRODUCTION

Alcoholism is considered a serious publichealth problem in Brazil (Burgos et al.,2004) that is more serious when occurringin pregnant and lactating women. Pregnantwomen are discouraged from drinkingalcohol because of detrimental effects onfetal development. Nonetheless, in manycultures, including in Brazil, lactatingwomen are encouraged to drink alcohol tooptimize breast milk production (Blume,1987; Mennella et al., 2005).

Ethanol (EtOH) (and possibly itsproducts) can pass from the maternalcirculation into breast milk. Mennella et al.(2005) have demonstrated that in humanbeings ethanol ingested through breast milkhas adverse effects on the child. This studyshowed that babies drank less milk and hada three-hour-disturbed sleep-wake patternafter their nursing mothers consumed one totwo standard drinks.

Studies using laboratory animals havedemonstrated that administering EtOH todrinking water during pregnancy, and even

AZARA ET AL. Biol Res 41, 2008, 317-330318

earlier in the mother’s life, can impairmammary gland development (Tavares doCarmo et al., 1996; Fuentes et al., 2001).These alterations can contribute to alteringthe production and nutritional compositionof the milk. In fact, administering EtOHduring pregnancy and lactation can alter themacronutrient composition of milk(Herrera, 1981; Heil et al., 1998; Tavaresdo Carmo et al., 1999; Albuquerque et al.,2000), and, therefore, the supply ofnutrients for the pups, as milk is the onlysource of nutrients during this period, withconsequences for their development. On theother hand, pregnancy and lactation areperiods of high mineral requirements andthere are few studies of the micronutrientstatus of milk, particularly of mineralsassociated with EtOH intake duringlactation.

However, despite extensive investigationof postnatal ethanol exposure usinglactating dams fed on ethanol liquid diets(Sanchis et al., 1989; Zhu and Seelig, 2000;Oyama and Oller Do Nascimento, 2003),there is a lack of information about thenutritional characteristics of milk,particularly micronutrients.

The present study sought to determinewhether EtOH administration in a liquiddiet during rat lactation affects the macroand micronutrient composition of the milk.The work was extended to determine brainand liver mineral status in suckling pups.The milk volume produced was alsoassessed in lactating rats.

2. MATERIALS AND METHODS

Animals

The Experimental Research Committeeof the Universidade Federal do Rio deJaneiro approved all procedures involvinganimals.

Adult virgin female Wistar rats weighing180 to 220 g, approximately 90 days of age,were obtained from the animal breedingunit of the Instituto de Nutrição Josué deCastro, Universidade Federal do Rio deJaneiro, Rio de Janeiro State, Brazil. Therats were mated to obtain the first offspring.

On day 1 of lactation, animals wererandomly divided into two groups: ethanol-fed group (EFG) n = 10 and pair-fed group(PFG) n= 10 and placed in individualmetabolic cages, on a 12-h light / 12-h darkcycle in a temperature and relativehumidity-controlled room (22ºC-26ºC and50%, respectively).

Experimental design

Rats were pair-fed from days 1-14 oflactation, receiving a nutritionally adequateLieber-DeCarli liquid diet formulated forthe lactating period requirements ofrodents, according to the American Instituteof Nutrition (AIN) (Reeves et al., 1993), asthe sole source of food (AIN 93G #710301for EFG and AIN 93G #710079 for PFG;Dyets Inc., Bethlehem, PA, USA) (Table I).Diets contained 4.2 kilojoule per milliliter(19.7% and 17.0% of total energy intake asprotein and fat, respectively). Microelementamounts in both diets were largely abovethe recommendations for lactation, thusensuring that the rats had sufficient intakeof vitamins and minerals. The EFGreceived the liquid diet ad libitum, butmaltose dextrose was substitutedisocalorically by ethanol to provide 34% ofthe total energy (Table I).

The PFG received the isocaloric liquiddiet without ethanol in a volume equal tothat ingested by rats of the EFG. Liquiddiets were renewed twice a day at 8:00 a.m.and 8:00 p.m., so the animals consumedethanol continuously until the time theywere killed. Daily fluid intake wasmeasured throughout the treatment period(days 1-14 of lactation). Dams wereweighed on days 0, 4, 8, and 12 of lactationand pups on days 1, 5, 10 and 14, everymorning at 08:00 a.m., on a digital balance(Coleman, PW- 1100 model).

Milk, blood, liver and brain collection

Milk samples were collected from dams onday 14 of lactation during the morning(Keen et al., 1981). After being separatedfrom their litters for 4 hours, they wereintraperitoneally injected with 5 units ofoxytocin (Sandoz Products Ltda), and after

319AZARA ET AL. Biol Res 41, 2008, 317-330

15 minutes were anesthetized withketamine (100 mg/Kg) and xylazine (16mg/Kg). The milk was obtained by gentlehand stripping of the teat. After milking,dams were decapitated and blood collectedin heparinized tubes. Plasma was obtainedby centrifugation and aliquots were used tomeasure maternal mineral levels. Pups weredecapitated; their brains and livers wereremoved and weighed, pooled for eachlitter, and then frozen in liquid nitrogen andkept at -80ºC until analysis.

Milk production and analysis

Milk yield was estimated from pup weightand weight gains on days 1-2, 7-8 and 12-13of lactation, as described previously(Sampson and Jansen, 1984). Milk sampleswere obtained from day 14 of lactation. Milk

samples were diluted 50 times with de-ionized water for the determination of milkcarbohydrate and protein. For lipid analysis,the dilution factor used was 10. For mineraland fatty acid determinations, integral milkwas used. Carbohydrate determination wasdone by colorimetric method described byDubois et al. (1956). Protein concentrationwas analyzed by the procedure of Lowry etal. (1951). The fat content of these sampleswas gravimetrically determined withpetroleum ether, according to the methoddescribed by Godbole et al. (1981).

The energy value of the milk wascalculated using the values of 4 Kcal/g forprotein and lactose, and 9 Kcal/g for fat.The milk nutrient output of day 14 wasestimated from the values of milkcomposition and corresponding milkproduction.

TABLE 1

Diet composition [grams per liter (g/L)]

Ingredient EFG (g/L) PFG (g/L)

Casein 53.00 53.00

L-cystine 0.80 0.80

Cellulose 13.30 13.30

Maltose dextrin 43.30 135.40

Ethanol (95%)* 65.40 —-

Sucrose 26.50 26.50

Soybean oil (stabilizer T-butylhydroquinone) 18.60 18.60

Salt mix a 9.28 9.28

Vitamin mixb 2.65 2.65

Choline bitartrate 0.53 0.53

Xanthan gum 3.00 3.00

* ethanol is in ml/L.a Contained the following (g/L): calcium carbonate = 357.00; potassium phosphate, monobasic = 196.00;potassium citrate, H2O = 70.78; sodium chloride = 74.00; potassium sulfate = 46.60; magnesium oxide =24.00; ferrous sulfate, 7 H2O = 5.21; zinc carbonate = 1.65; manganous carbonate = 0.63; cupric carbonate =0.30; potassium iodate = 0.01; sodium selenate = 0.01025; ammonium paramolybdate, 4 H2O = 0.00795;sodiummetasilicate, 9 H2O = 1.45; chromium potassium sulfate, 12 H2O = 0.275; lithium chloride = 0.0174;boric acid = 0.0815; sodium fluoride = 0.0635; nickel carbonate = 0.0318; ammonium vanade = 0.0066; andsucrose, finely powdered = 221.87.b Contained the following (g/L): niacin = 3.00; calcium pantothenate = 1.60; pyridoxine hydrochloride = 0.70;thiamine hydrochloride = 0.60; riboflavin = 0.60; folic acid = 0.20; biotin = 0.02; vitamin E acetate (500 IU/g) = 15.00; vitamin B12 (0.1%) = 2.50; vitamin A palm (500.000 IU/g) = 0.80; vitamin D3 (400.000 IU/g) =0.25; vitamin K1 premix (10 mg/g) = 7.50; and sucrose = 967.23.


Fatty acid determination

Lipid extraction, saponification, andmethylation of fatty acids in milk wereperformed in duplicate from a 200 μLsample of milk according to the method ofLepage and Roy (1986), which recommendstreatment with 2 mL of 4:1 (v/v)methanol:benzene solution, and addition of200 μL of acetyl chloride under lightagitation. Fatty acid methyl esters weremeasured in a Perkin Elmerchromatography autosystem provided witha hydrogen flame ionization detector and acapillary column (60m x 0.30mm i.d.),packed with 10% SP 2330 (Supelco Inc,Bellefonte, PA, USA) as a stationary phase.Nitrogen was used as a carrier gas.Injection and detection temperatures wereboth at 220ºC, whereas oven temperaturewas programmed to vary 5°C/min from 40to 225°C. Esters were identified comparingretention times with known standards(Sigma Aldrich, St Louis, MO, USA), andquantification was done by calculating peakareas with an integrator. Results wereexpressed as a weight percentage (g/100 gtotal fatty acids).

Mineral determination

The levels of minerals were determinedby total reflection X-ray fluorescenceanalysis (TXRF) with synchrotronradiation. The samples (milk, brain, liverand plasma) were thawed and homogenized,using a vortex shaker prior to sub sampling.0.5mL of each sample was transferred topolyethylene vials free of trace elements,and treated with 0.5mL of HNO3 and 0.5mLof H2O2 at 110ºC for 24 hours, with thevials sealed. After this, the solution was leftto dry and the volume recovered to 0.5mLwith a solution of HNO3 0.1M as a dilutingagent. A 50 mL of solution (40 mg/g Ga) ofgallium (Ga)F was added to the sample,which was used as an internal standard ofreference for the TXRF measurements inorder to obtain a concentration of 10 ppmof Ga in the solution. A 5 mL aliquot of thesolution was placed on a Perspex samplesupport using a pipette, and subsequentlysubmitted to drying in a vacuum dryer for 3

hours, obtaining spots of approximately5mm in diameter. Measurement of thesamples by TXRF was carried out at theNational Laboratory of Synchrotron Light(LNLS), Campinas, São Paulo State, Brazil.All samples were processed in duplicate,and the final values were taken as themeans of the results, obtained by runningeach sample three times. A white beam ofirradiation with a maximum energy of 20keV altered by 0.5mm of aluminum, withan angle of incidence of 1.0 mrad, wasutilized to excite the sample. Thecharacteristic X rays were detected by asilicon-lithium (Si-Li) detector with aresolution of 165 eV for 5.9 keV energy,linked to an electronic system with amultichannel analyzer (Canberra). Thedistance between the detector and samplewas fixed at 6.0 mm, using a tantalum (Ta)collimator with an aperture of 1.0 mm inorder to limit the dead time of themeasurements to a minimum value of 15%.The spectrometer sensitivity wasdetermined using multi-element standardswith different concentrations containing Al,Si, Ca, Ti, Cr, Fe, Ni, Zn, Ga, and Se. Thestandards were prepared from mono-elemental solutions (induced coupledplasma (ICP), supplied by Sigma (St. Louis,MO, USA). The accuracy of themeasurements was calculated bydetermining the concentration of theelements of a standard solution (ICP-multi-element standard solution FMERCK). Themeasurement time was 300 s for thesamples and 150 s for the standards. Thespectrums were analyzed by a quantitativeanalysis program (quantitative X-rayanalysis system FQXAS) distributed by theInternational Atomic Energy Agency(IAEA), which gives the fluorescent countintensities for each element and theassociated uncertainty (Klockenkämper,1992).

Statistical analysis

Results were expressed as mean ± SEM.Differences between means were tested forsignificance by Student’s t test. Differencesbetween groups were considered statisticallysignificant when p<0.05 or p< 0.001.


3. RESULTS

Alcohol intake and body mass change ofdams

In the present study, EtOH wasadministered in the liquid diet for 14 days(postnatal days 1-14) of lactating rats,resulting in a daily ethanol intake ofbetween 12.8 and 14.9 g/Kg, and a bloodethanol concentration of 25mM (data notshown). Dams looked healthy and gainedweight throughout the feeding period. Thecorporal mass of the mothers showed nosignificant difference (p>0.05) betweenEFG and its respective PFG during the first14 days of lactation (data not shown).

Milk production and pup weight gain

Figure 1A shows the milk production of thefemales of EFG and PFG during days 1-2,6-7, 9-10, and 13-14 of lactation. After day7 until day 14 of lactation, the lactatingfemale rats that ingested ethanol had alower production of milk in relation to thevalues of PFG (p<0.001). The values ofmilk production on day 14 of lactation were2.42 ± 0.2 mL/day from PFG, and 1.76 ±0.01 mL/day from EFG (p<0.001). Thebody mass of pups nursed by the twogroups of dams is shown in Figure 1B.From day 11 on, the pups from the EFG didnot maintain the same profile of bodyweight gain compared to those from thePFG. The reduced milk production ofethanol-treated mothers was associated witha significant reduction in corporal massgain of their pups and these differencesbecame more evident as the lactating periodadvanced (Figure 1B).

Milk macronutrient composition

Milk composition and the estimated milkmacronutrient output on day 14 of the twoexperimental groups are shown in Table II.Ethanol-treated mothers produced milk withsignificantly less carbohydrates and moreprotein concentrations (p<0.05), whereasthe lipid content was unaffected.

The macronutr ients were alsocalculated in agreement with the

production of milk for 24 hours on days12-14 of lactation, characterized as thepeak period of lactation. We observed asmaller concentration of proteins andcarbohydrates and a greater concentrationof lipids in the EFG milk compared to thatof the PFG group, with signif icantdifference (p<0.05).

It was also observed that the energeticvalue of the EFG milk presented valuessignificantly higher (p<0.05).

Regarding the fatty acid profile in dammilk on day 14 of lactation, the proportionof medium-chain fatty acids (C10:0 andC12:0), C18:0, 16:1 n-7 and 18:1 n-9 inmilk was significantly higher in EFG thanin PFG. The proportion of other fattyacids, including linoleic acid (18:2 n-6)and a-linolenic acid (18:3 n-3), was similarin both groups. Futhermore, a lowproport ion of DHA (22:6 n-3) wasobserved in milk from ethanol-treateddams as compared to that of the PFG(p<0.05). (Table III).

Milk micronutrient composition

The effects of maternal ethanol intake onmilk mineral concentration are presented inTable IV. The ethanol-treated mothersproduced milk with increasedconcentrations of several minerals, such asCa (64%), P (45%), S (42%), K (149 %), Cl(227 %), Zn (38.8 %), Cu (91%), Sr (27%),Cr (76%), Mn (80%), Se (152%), Ni (56%)in relation to PFG (p<0.05 or p<0.001). Themilk Fe levels were similar in both groupsstudied. When the concentrations of theseminerals present in the milk output on day14 of lactation were examined, we observedthat all minerals were similar in quantitiesbetween the groups, but Cl and Sedisplayed values 114% and 86% higher inEFG compared to PFG.

Plasma mineral concentration in dams andpups

Table V presents the averageconcentration of minerals in the plasma ofmothers and pups of both groups .Phosphate (P) and ferrous (Fe) displayedlower concentrations, and bromine (Br)


Figure 1: Effect of ethanol intake on milk production and pup body mass during 14 days oflactation. The production of milk was determined in controls and ethanol-treated female ratsaccording to the technique described by Sampson and Jansen (1984). Milk production (Panel A)and pup body mass (Panel B) from each group were estimated as described in the Materials andMethods section. The symbols are -O- PFG; -•- EFG. Values are mean ± SEM from triplicate atthree independent experiments with 8-10 animals per group. * Significantly different at p<0.05 and** significantly different at p<0.001.


TABLE 2

Effect of ethanol intake by lactating rats on milk macronutrient composition (expressed bymL or day production) and energetic value (Kcal/100 mL).

The values were obtained from the milk of day 14 of lactation.

Macronutrients/ Milk (mL production) (μg/mL) Milk (day production) (μg/day)

Energetic value C EtOH C EtOH

Protein 12.17±0.12(23.4%) 14.53±0.17**(28.1%) 29.46±0.29(28.9%) 25.57±0.31**(23.1%)

Lipid 16.14±0.71(69.8%) 15.31±0.41(66.7%) 28.41±0.9(62.8%) 36.97±1.25**(72.6%)

Carbohydrate 3.51±0.09(6.8%) 2.73±0.05**(5.2%) 8.50±0.21(8.3%) 4.80±0.10**(4.3%)

Energetic value(Kcal/100mL) 202.90±3.22 212.10±3.76 407.10±10.86 459.70±8.53*

Values are mean ± SEM of 8-10 samples. EtOH- Ethanol-fed group; C - Pair-fed group.* Significantly different from C at p<0.05.** Significantly different from C at p<0.001.

showed higher concentrat ions in theplasma of EFG dams compared to that ofPFG. Despi te these d i f ferences , theconcentrat ion of other minerals wass imi lar in both groups of dams. Insuckl ing pups f rom ethanol- t rea tedmothers a lower concentration of Cu andSr was observed in plasma (Table V). Theother minerals did not present significantchange.

Liver and brain mineral status of pups

As observed in Table VI, the mineralconcentrations in the liver did not differbetween the two groups. In the brain, weobserved that Ca and Cl concentrations weresignificantly lower in pups from ethanol-treated mothers compared to those fromPFG. Moreover, a higher concentration ofCd was observed in the brain from EFG.

TABLE 3

Composition percentage (%) of fatty acids in the milk of dams treated with ethanol andcontrol diet in 14-day-old pups

Fatty acids C EtOH

C 10:0 2.82 ± 0.09 4.17 ± 0.12**

C 12:0 4.04 ± 0.01 6.80 ± 0.06**

C 14:0 18.90 ± 1.03 19.48 ± 1.08

C 16:0 23.25 ± 1.85 20.75 ± 1.04

C 18:0 3.87 ± 0.36 5.30 ± 0.48*

C 16:1 n-7 0.97 ± 0.03 1.77 ± 0.08*

C 18:1 n-9 17.95 ± 1.02 21.05 ± 0.91*

C 18:2 n-6 19.06 ± 1.25 18.02 ± 1.69

C 18:3 n-3 1.04 ± 0.04 1.06 ± 0.08

C 20:4 (ω-6)AA 1.13 ± 0.07 1.12 ± 0.09

C 22:6 (ω-3)DHA 1.03 ± 0.08 0.55 ± 0.01*

Values are mean ± SEM of 8 samples. EtOH- Ethanol-fed group; C - Pair-fed group AA, arachidonic acid;DHA - Docosahexaenoic acid.* Significantly different from C at p<0.05.** Significantly different from C at p<0.001.


TABLE 4

Effect of ethanol intake by lactating rats on milk mineral concentration on day 14of lactation

Minerals milk (μg/mL production) milk (μg/day production)

C EtOH C EtOH

Ca 2915.71 ± 245.08 4793.68 ± 419.86* 7055.88 ± 593.13 8438.95 ± 738.54

P 543.93 ± 30.15 790.74 ± 62.44* 1303.56 ± 94.33 1310.71 ± 113.49

S 542.00 ± 36.28 768.66 ± 53.74* 1311.58 ± 87.79 1352.81 ± 94.57

K 192.67 ± 18.92 480.04 ± 46.18* 661.84 ± 60.24 844.56 ± 81.25

Cl 30.46 ± 2.26 99.68 ± 9.94** 78.52 ± 6.74 175.65 ± 17.51**

Zn 10.06 ± 0.70 13.96 ± 1.12* 21.88 ± 0.51 24.55 ± 1.98

Fe 5.81 ± 0.37 6.97 ± 0.33 14.07 ± 0.91 12.26 ± 0.58

Cu 1.84 ± 0.17 3.52 ± 0.15** 5.38 ± 0.53 6.19 ± 0.27

Sr 0.62 ± 0.04 0.79 ± 0.07* 1.50 ± 0.10 1.39 ± 0.12

Cr 0.25 ± 0.02 0.44 ± 0.01** 0.62 ± 0.06 0.78 ± 0.02

Mn 0.20 ± 0.01 0.36 ± 0.03* 0.59 ± 0.05 0.63 ± 0.05

Se 0.03 ± 0.00 0.08 ± 0.00** 0.07 ± 0.00 0.14 ± 0.00**

Ni 0.09 ± 0.01 0.14 ± 0.01* 0.19 ± 0.00 0.24 ± 0.01

Values are mean ± SEM of 6-8 samples. EtOH- Ethanol-fed group; C - Pair-fed group.* Significantly different from C at p<0.05.** Significantly different from C at p<0.001.

TABLE 5

Effect of ethanol intake by lactating rats on the plasma mineral concentration on pupsand dams on day 14 of lactation

Minerals dams (μg/mL) pups (μg/mL)

C EtOH C EtOH

Ca 89.97 ± 13.78 76.01 ± 4.03 87.86 ± 2.96 90.40 ± 2.69P 58.23 ± 3.23 44.67 ± 4.52** 110.48 ± 6.32 110.22 ± 6.03S 553.52 ± 28.30 516.78 ± 24.86 494.03 ± 14.69 459.83 ± 9.96K 179.77 ± 12.16 160.30 ± 9.82 289.39”± 23.48 282.85 ± 23.31Cl 1462.86 ± 62.76 1478.29 ± 50.79 1742.52 ± 94.05 1521.06 ± 61.10Zn 1.52 ± 0.11 1.49 ± 0.05 2.32 ± 0.06 2.42 ± 0.09Fe 4.30 ± 0.39 3.04 ± 0.24* 1.95 ± 0.16 2.72 ± 0.29Cu 0.97 ± 0.03 1.22 ± 0.18 0.46 ± 0.03 0.33 ± 0.01*Sr 0.17 ± 0.01 0.16 ± 0.02 0.07 ± .0.01 0.05 ± 0.01**Cr ND ND ND NDMn 0.059 ± 0.00 0.06 ± 0.00 0.05 ± 0.00 0.07 ± 0.01Se 0.24 ± 0.01 0.23 ± 0.01 0.10 ± 0.01 0.09 ± 0.01Br 1.59 ± 0.04 1.90 ± 0.02* 3.40 ± 0.18 3.42 ± 0.15

Ni 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00

Values are mean ± SEM of 6-8 samples. EtOH- Ethanol-fed group; C - Pair-fed group.ND – Not determined* Significantly different from C at p<0.05.** Significantly different from C at p<0.001.


DISCUSSION

In the present study, EtOH wasadministered in the liquid diet for 14 days(postnatal days 1-14) to lactating rats,resulting in a daily ethanol intake between12.8 and 14.9 g/Kg, and a blood ethanolconcentration of 25mM (data not shown).In addition, the analysis of maternal bodyweight has not revealed significantdifferences between the EFG and the PFG,demonstrating that ethanol intake in thiswork has not resulted in nutritionalalterations that could lead to changes in theweight gain of lactating rats. Besides, ratsfrom both groups ingested energy andnutrient amounts within the nutritionalrequirements for lactating rats, according tothe AIN (Reeves et al., 1993). Peres et al.(2004), administering alcoholic liquid dietduring gestation, showed that there was nosignificant difference between the groupsregarding dietary consumption and bodyweight during the whole experimentalperiod. Thus, based on the availableliterature, and also according to our results

of ethanol intake and nutritional status ofmothers, the experimental model of thepresent study showed that the deleteriouseffects of ethanol intake are not derivedfrom the malnutrition caused by it.

It is known that after 30 minutes ofethanol ingestion it is already present in thematernal milk and it modifies the sensorialcharacteristics of the milk even in lowconcentrations (Mennella and Beauchamp,1993, Subramanian et al., 1999, Lima et al.,2002; Burgos et al., 2004). This processalso leads to a reduced suction of the pupsand consequent decrease of the hormonelevels responsible for the synthesis andejection of the milk (Tucker, 1979; Vilaróet al . , 1987; Whitworth, 1988;Subramanian, 1999). Analyzing the resultsof Figure 1A, we can conclude that theethanol intake caused a reduction in milkproduction, starting on day 7 of lactation.According to the literature cited, it wasprobably due to a decrease in the levels ofthe hormones prolactin and oxytocin,responsible, respectively, for the synthesisand ejection of the milk. On the other hand,

TABLE 6

Effect of ethanol intake by lactating rats on the liver and brain mineral concentration(mg/g) of pups on day 14 of lactation

Minerals liver (μg/g) brain (μg/g)

PFG EFG PFG EFG

Ca 0.19 ± 0.003 0.18 ± 0.016 3.42 ± 0.370 1.07 ± 0.550*

P 3.12 ± 0.002 3.03 ± 0.001 8.65 ± 1.110 4.56 ± 0.140*

S 2.44 ± 0.003 2.53 ± 0.003 4.53 ± 0.580 3.46 ± 0.120

Cl 0.73 ± 0.005 0.70 ± 0.001 1.88 ± 0.230 1.18 ± 0.050*

Zn 0.04 ± 0.005 0.03 ± 0.001 0.05 ± 0.007 0.03 ± 0.003

Fe 0.05 ± 0.002 0.06 ± 0.006 0.11 ± 0.010 0.06 ± 0.003

Cu 0.04 ± 0.003 0.03 ± 0.003 0.02 ± 0.004 0.01± 0.001

Sr ND ND ND ND

Cr 0.01 ± 0.001 0.01 ± 0.001 0.03 ± 0.010 0.03 ± 0.010

Mn 0.03 ± 0.002 0.03 ± 0.002 0.07 ± 0.060 0.04 ± 0.005

Se 0.01 ± 0.002 0.01 ± 0.001 0.02 ± 0.005 0.01 ± 0.005

Cd 2.44 ± 0.111 2.36 ± 0.002 2.30 ± 0.230 3.83 ± 0.230**

Values are mean ± SEM of 6-8 samples. EFG- Ethanol-fed group; PFG - Pair-fed group.ND – Not determined* Significantly different from PFG at p<0.05.** Significantly different from PFG at p<0.001.


probably, part of this effect relates to theendocrine effects of ethanol consumptionthat involves inhibition of antidiuretichormone release, thus impairing the waterbalance with changes in milk volume andcomposition, although these effects aremore frequent when ethanol was added tothe drinking fluid (Jones et al., 1981;Marquis et al., 1984; Testar et al.,1986).

In addition, it is not possible in mostexperiments to separate the effects ofalcohol from those caused by its metabolicproduct, acetaldehyde. Thus, whether or notthese effects of alcohol impairing milkproduction could be caused by acetaldehydeinstead of ethanol is a subject for moreinvestigations, since there were no adequatereports found in the literature. In lactatingrats, Guerri and Sanchis (1986) haveobserved that acetaldehyde levels in milkwere always 35-45% lower than in blood,and Kesaniemi (1974), with lactatingwomen, showed that acetaldehyde, the toxicmetabolite of alcohol, does not appear topass to breast milk.

In relation to alteration in milkproduction in EFG, we observed a decreasein pup growth, mostly at the lactation peak(days 12-14 of lactation). This is themaximum milk production period in rats(Figure 1B), suggesting that this effectmight be a consequence of the ethanolintake by the dams, producing less milkthan their capacity and not matching themilk requirements of the pups, thusimpairing pup growth. These results agreewith those of other investigators using otherexperimental models for administration ofethanol during pregnancy and/or lactation(Vilaró et al., 1987; Tavares do Carmo etal., 1999; Heil and Subramanian, 2000).

Rat pups (Molina et al., 2007) andhuman infants (Faas et al., 2000; Mennellaand Beauchamp, 1993; Mennella, 1997)seem capable of processing ethanol presentin the milk during the nursing period. Inanother study, Pueta et al, 2008 observedthat intoxication during nursing disruptedthe capability of the dam to retrieve thepups and to adopt a crouching posture. Onthe other hand, the consumption of a singledose of alcoholic beer by nursing mothersflavored their milk and decreased the

amount of milk consumed by their infants(Mennella and Beauchamp, 1993). All thesefactors may have functional consequencesfor milk yield and milk composition, with anegative impact on growth rates ofoffspring.

In addition to the effect of alcoholimpairing milk production, we observedthat ethanol in the liquid diet also affectedmilk composition. When compared to PFG,EFG presented similar milk lipidconcentrations, with significantly lesscarbohydrates and higher proteinconcentration. Vilaró et al. (1987) observeddecreased lactose and increased triglycerideconcentration in milk of ethanol-treatedrats. Brigham et al. (1992), using anotherexperimental model, suggested that this isan expected finding because carbohydrateconcentration is positively associated withmilk volume, whereas protein concentrationis not. Aditionally, the possibility thatethanol can reduce glucose productionthrough the gluconeogenic pathway cannotbe discarded. This could contribute todiminishing the carbohydrate abundance ofmilk (Sumida et al., 2007). Finally, it isalso possible that lower carbohydrateconcentration in the EFG milk reflects thereduction of lactose synthesis in themammary gland, due to a probabledeviation of the carbohydrates for lipidsynthesis in this tissue. This hypothesisseems to be valid, since other studies haveshown that maternal intake of ethanolduring the first 12 days of lactationenhanced the mammary gland lipogenesis(Tavares do Carmo et al., 1996), andenhanced the mammary gland uptake ofcirculating fatty acid triglycerides due toaugmented lipoprotein lipase activity(Vilaró et al., 1987). The fatty acid profilein milk of ethanol-treated rats in this studymay also be a consequence of the effectsshown by these authors. The proportion ofmedium-chain fatty acids (C10:0 andC12:0) was higher in EFG when comparedto PFG, which would indicate a specificabundance of these lipogenic products inthe mammary gland of ethanol-treated rats.Since it is known that triglyceridescontaining medium-chain fatty acids aremore easily hydrolyzed and absorbed


during suckling than long-chain fatty acids(Neville and Picciano, 1997; Genzel-Boroviczény et al., 1997), the preservationof those fatty acids in milk of ethanol-treated lactating mothers may well be ametabolic adaptation addressed to guaranteeoffspring survival, even when the amountof available milk is not sufficient.According to this, although the ethanol-treated dams produced less milk thancontrols, the volume measurement and themacronutrient analysis of the milk on day14 resulted in a higher energy content in themilk of the ethanol-treated rats (Table II),this being possible because the energeticvalue of lipids is greater than that of proteinand carbohydrates.

DHA is essential for the growth andfunctional development of the brain ininfants (Marszalek and Lodish, 2005). Theproportional increments in C18:1, C16:1and C18:0, and the decrease in C22:6 n-3 inmilk found in the present study in the EFGare probably the result of ethanol action onfatty acid metabolism. Ethanol is known toinhibit both Δ6-desaturase and Δ5-desaturase activities (Nervi et al., 1980)which are responsible for the conversion ofdietary linoleic acid (18:2 n-6) and alpha-linolenic (18:3 n-3) to their respectivemetabolites, arachidonic acid (20:4 n-6) anddocosahexaenoic acid (DHA, 22:6n-3).Thus, these inhibitory effects of ethanol onthe activity of these enzymes may beresponsible for the decrease in the C22:6 n-3 fatty acid found here in milk of theethanol-treated lactating rats.

To our knowledge, this is the first reportof the profile of several minerals in milkfrom lactating rats receiving ethanol inliquid diets. In the present study, formineral analysis, we used the TotalReflection X-Ray Fluorescence (TXRF)technique. This is an extremely useful toolin mineral determination. Its mainadvantages over other methods are the lowdetection limits and the simultaneousdetermination of several elements,employing small amounts of sample(Klockenkämper, 1992). This lattercharacteristic is extremely importantconsidering the small amount of milk thatcan be collected from rats in each

experiment (1-2mL/rat). The procedureused for measuring the mineral content ofthe rat milk samples provided informationon the total quantity of minerals. We couldnot partit ion the ions between theirdiffusible and non-diffusible pools. Theresults reported can therefore only representthe total ion content of the milk.

Present results show that ethanoltreatment affects milk mineral composition.Ethanol-treated mothers presented anincrease in the concentration of severalminerals, such as Ca, P, S, K, Cl, Zn, Cu, Sr,Cr, Mn, Se, and Ni in relation to PFG (TableIV). These results suggest an adaptativeincrease in some elements. On the otherhand, the mechanisms of the divergentresponses between EFG and PFG diets couldnot be ascertained from the present results.The possible hypothesis would be: (a) Sincemilk salts exist primarily as either ions insolution, bound to protein or associated withcasein micelles, they are concentrated in themilk aqueous phase, and (b) Variations inthe milk lipid content will therefore dilutethe ion concentration in whole milk andpossibly mask any changes in ionconcentration associated with the aqueousphase. It is therefore worth considering thepossible effects of ethanol intake on the ionconcentration of fat-free milk (FFM) infuture studies.

Another explanation for this increase inthe mineral concentration in the EFG milkwould be an increase of bioavailability of atrace element for the mammary gland ofEFG (probably due to the high mobilizationfrom other tissues) that can influence itsactual concentration in milk. To confirmthis hypothesis, future works in this fieldwill be necessary.

When we examined the concentrationsof these minerals by milk output on day 14of lactation, we observed that all mineralswere similar in quantities between thegroups, except for Cl and Se, whichpresented higher levels in EFG compared toPFG. This also corresponds to the adequatelevel of circulating minerals we found inthe mothers, with the exception of P and Fethat were reduced (Table V).

Some minerals have been implicated inthe development of the neonate, and several


complications in newborns, such asdeficiency in the growth and intellectualdevelopment that have been associated withthe deficiency of iron, copper and zinc(Haschke et al., 1995; Costa et al., 2002).

In the rat during suckling, hepatic storesthat were accumulated in the uterus are asubstantial source of minerals, mostly Cu+2,for neonates (Keen et al., 1981). This studyhas revealed lower concentration of Cu+2 inthe plasma of suckling pups from ethanol-treated mothers (Table V), while themineral concentrations in the liver do notdiffer between the groups (Table VI).Burmistrov and Borodkin (1990) andJacques et al. (1989) also observed thatserum copper levels decrease with alcoholintake. Since Cu+2 or ceruloplasmin isprogressively unloaded from the liver andreleased into the blood during neonataldevelopment (Prohaska, 1990) and we havenot observed a decrease of Cu+2 in liver inthe present study, it is possible that ethanolalters the pattern of Cu+2 (orcerulosplasmin) release into the blood. Thelower levels of Cu+2 in plasma of pups fromEFG could also be attributed to an inabilityto synthesize the protein cerulosplasminrather than to a lack of available Cu+2, sincea high percentage of the copper circulatingin plasma is bound to this protein(Hambidge, 2003). This hypothesis agreeswith the observation that ethanol treatmentdecreased hepatic synthesis and thesecretion of proteins.

The present study also showed that thebrains of the litters of ethanol-treatedmothers presented lower concentrations ofCa and Cl, and higher concentration of Cd.It is known that brain cells, particularlyneurons, are highly dependent on properamounts of available sodium, potassium,chloride, and calcium. Therefore anydisruption in the proper flow andavailability of these electrolytes alters thefunctionality of the neurons, leading tomodifications both in behavior and in theability of the brain to regulate other organicprocesses (Bourre, 2004). On the otherhand, cadmium is one of the most toxicheavy metals (Brzóska et al., 2002) andethanol is able to increase the permeabilityof biological membranes to various

substances, including toxic metals (Pal etal., 1993), and was reported to increase Cdretention in rat brain (Pal et al., 1993;Brzóska et al., 2000). Brzóska et al. (2002)showed that Cd caused disturbances in Znand Cu metabolism, reflected by changes inthese bioelement concentrations andcontents in tissues and biological fluids.

All these alterations on brain mineralcomposition in the EFG pups occur duringa sensitive brain development period andmay lead to an abnormal development, withpermanent effects over life. Therefore, theresults of the current study – showing theeffects produced by ethanol during lactationon lactating performance and mineral statusof pups æ must be taken into account whenadvising mothers about alcohol intakeduring lactation.

ACKNOWLEDGEMENTS

This work was supported by grants fromFundação Universitária José Bonifácio(FUJB), Conselho Nacional deDesenvolvimento Científico e Tecnológico(CNPq), and Fundação de Amparo àPesquisa Carlos Chagas Filho do Estado doRio de Janeiro (FAPERJ).

REFERENCES

ALBUQUERQUE KT, TAVARES DO CARMO MG,HERRERA E. 2000. Ethanol Consumption byLactating Rats Induces Changes in Pup’s Fatty AcidProfiles. Nutr. Neurosci. 3: 331-337

BLUME, S. 1987. Beer and breast-feeding mom. J. Amer.Assoc. 15: 21-26

BOURRE JM. 2004. The role of nutritional factors on thestructure and function of the brain: an update ondietary requirements. Rev. Neurol. 160 (8-9): 767-792

BRIGHAM HE, SAKANASHI TM, RASMUSSEN KM.1992. The effect of food restriction during thereproductive cycle on organ growth and milk yield andcomposition in the rat. Nutrition Research 12: 845-856

BRZÓSKA MM, MONIUSZKO-JAKONIUK J, JUECZUKM, GALAZYN-SIDORCZUK M. 2002. Cadmiumturnover and changes of zinc and copper body status ofrats continuously exposed to cadmium and ethanol.Alcohol and Alcoholism 37(3): 213-221

BRZÓSKA MM, MONIUSZKO-JAKONIUK J, JURCZUKM, GALAZYN-SIDORCZUK M, ROGALSKA J.2000. Effect of short-term ethanol administration oncadmium retention and bioelements metabolism in ratscontinuously exposed to cadmium. Alcohol andAlcoholism 35: 439-445

BURGOS, MGPA, BION FM, CAMPOS F. 2004. Lactação


e álcool: Efeitos clínicos e nutricionais. ArchivosLatinoamericanos de Nutricion 54 (1): 25-35

BURMISTROV SO, BORODKIN IUS. 1990. Thecharacteristics of the enzyme status of the antioxidantprotection and the level of lipid peroxidation in thebrain tissue and blood of rats with differing preferencesfor ethanol. Farmakol. Toksikol. 53(5): 59-60

COSTA RS, TAVARES DO CARMO MG, SAUNDERS C,LOPES RT, DE JESUS EF, SIMABUCO SM. 2002.Trace elements content of colostrum milk in Brazil.Journal of Food Composition and Analysis 15: 27-33

DUBOIS M, GILLES KA, HAMILTON J, REBERS PA,SMITH F. 1956. Colorimetric method fordetermination of sugars and related substances.Analytical Chemistry 28(3): 350-556

FAAS AE, SPONTÓN ED, MOYA PR, MOLINA JC.2000. Differential responsiveness to alcohol odor inhuman neonates: effects of maternal consumptionduring gestation. Alcohol 22: 7-17

FUENTES LM, ARTILLO R, CARRERAS O. 2001.Effects of maternal chronic ethanol administration inthe rat: lactation performance and pup’s growth.European Journal Nutrition 40: 147-154

GENZEL-BOROVICZÉNY O, WAHLE J, KOLETZKO B.1997. Fatty acid composition of human milk during the1st month after term and preterm delivery Eur. J.Pediatr. 156(2): 142-147

GODBOLE VY, GRUNDLEGER ML, PASQUIME, TA,THENEN SW. 1981. Composition of rat milk from day5 to 20 of lactation and milk intake of lean andpreobese Zucker pups. J. Nutr. 111: 480-487

GUERRI C, SANCHIS R. 1986. Alcohol and acetaldeydein rat’s milk folowing administration. Life Sciences38(17): 1543-1556

HAMBIDGE M. 2003. Biomarkers of Trace Mineral Intakeand Status. J. Nutr. 133: 948S-955S

HASCHKE F, HUEMER C, PIETSCHNIG B. 1995. Traceelement deficiencies. In: Clinical Nutrition on theYoung Child. Nestec Ltd Vevey, Raven Press, NewYork, p. 547-560

HEIL SH, HUNGUND BL, ZHENG ZH, JEN KLC,SUBRAMANIAN MG. 1998. Ethanol and lactation:Effects on milk lipids and serum constituents. Alcohol18: 43-48

HEIL SH, SUBRAMANIAN MG. 2000. Chronic alcoholexposure and lactation: Extended observations. Alcohol21(2): 127-132

HERRERA E. 1981. Ethanol toxicity: Metabolic effectsand experimental model for the “fetal alcoholsyndrome”. General Pharmacology: The VascularSystem 12 (2): A17

JACQUES PF, SULSKY S, HARTZ SC, RUSSEL RM.1989. Moderate alcohol intake and nutritional status innonalcoholic elderly subjects. Am. J. Clin. Nutr. 50:875-883

JONES PJ, LEICHTER J, LEE M. 1981. Placental bloodflow in rats fed alcohol before and during gestation.Life Sci. 29(11): 1153-9

KEEN CL, LONNERDAL B, CLEGG M, HURLEY L.1981. Developmental changes in composition of ratmilk: trace elements, minerals protein, carbohydrateand fat. Journal of Nutrition 111 (2): 226-230

KESANIEMI YA. Ethanol and acetaldehyde in the milkand peripheral blood of lactating women after ethanoladministration. 1974. J Obstet Gynaecol Br Commonw81(1): 84-6

KLOCKENKÄMPER R. 1992. Total reflection x-rayfluorescence spectroscopy. Analytical Chemistry 64,1115A

LEPAGE G, ROY CC. 1986. Direct transesterification of

all classes of lipid in one-step reaction. J. Lip. Res. 27:114-120

LIMA LA, MELO-JUNIOR MR, CAVALCANTI CLB,MELO CB, PONTES-FILHO NT. 2002 Does theexposition to alcohol in pre and post-natal periodsinterfere on the formation and maturation of PeyersPatches? An. Fac. Med. UFPE 47(1): 22-26

LOWRY OH, ROSEBROUGHT NJ, FARR AL,RANDALL RJ. 1951. Protein measurement with theFolin-Phenol reagent. J. Biol. Chem. 193: 265-275

MARQUIS SM, LEICTER J, LEE M. 1984. Plasma aminoacids and glucose levels in the rat fetus and dam afterchronic maternal alcohol consumption. Biol Neonate.46(1): 36-43

MARSZALEK JR, LODISH HF. 2005. Docosahexaenoicacid, fatty acid-interacting proteins, and neuronalfunction: Breastmilk and Fish Are Good for You. Ann.Rev. Cell. Dev. Biol. 21: 633-657

MENNELLA JA. 1997. Infant’s suckling responses to theflavor of alcohol in mother’s milk. Alcohol Clin ExpRes 21: 581-5

MENNELLA JA, BEAUCHAMP K. 1993. Effects of beeron breast-fed. J. Amer. Med. Assoc. 269: 1673-1678

MENNELLA JA, PEPINO MY, TEFF KL. 2005 Acutealcohol consumption disrupts the hormonal milieu oflactating women. J. Clin. Endocrinol. Metab. 90(4):1979-1985

MOLINA JC, SPEAR NE, SPEAR LP, MENNELLA JA,LEWIS MJ. 2007. Alcohol and development: BeyondFetal Alcohol Syndrome. Dev Psychobiol 49: 227-42

NERVI AM, PELUFFO RO, BRENNER RR, LEIKEN AI.1980 Effect of ethanol administration on fatty aciddesaturation. Lipids 15, 263-268

NEVILLE MC, PICCIANO MF. 1997. Regulation of milklipid secretion and composition. Ann. Rev. Nutr. 17:159-184

OYAMA LM; OLLER DO NASCIMENTO CM. 2003.Effect of ethanol intake during lactation on male andfemale pups’ liver and brain metabolism during thesuckling-weaning transition period. Nutr. Neurosci.6(3): 183-188

PAL R, NATH, R, GILL, KD. 1993. Influence of ethanolon cadmium accumulation and its impact on lipidperoxidation and membrane bound functional enzymes(Na+,K+-ATPase and acetylcholinesterase) in variousregions of adult rat brain. Neurochemical Investigation23: 451-458

PERES WAF, TAVARES DO CARMO MG, IGLESIASAC, ZUCOLOTO S, BRAULIO V. 2004. Ethanolintake inhibits growth of the epithelium in the intestineof pregnant rats. Alcohol 1: 5-9

PROHASKA JR. 1990. Development of copper deficiencyin neonatal mice. J. Nutr. Biochem. 1: 415-419

PUETA M, ABATE P, HAYMAL OB, SPEAR NE,MOLINA JC. 2008. Ethanol exposure during lategestation and nursing in the rat: Effects upon maternalcare, ethanol metabolism and infantile milk intake.Pharmacol Biochem Behav (2008), doi: 10.1016/j.pbb.2008.06.007 (in press)

REEVES PG, NIELSEN FH, FAHEY GC. 1993. AIN-93Purified diets for laboratory rodents: final report ofthe American institute of nutrition ad hoc writingcommittee on the reformulation of the AIN-75Arodent diet. American Institute of Nutrition 22: 3166-3193

SAMPSON, DA, JANSEN, GR. 1984. Measurement ofmilk yield in the lactating rat from pup weight andweight gain. J. Pediatr. Gastroenterol. Nutr. 3: 613-617

SANCHIS R, SANCHO-TELLO M, GUERRI C. 1989. Therole of liquid diet formulation in the postnatal ethanol


exposure of rats via mother’s milk. J. Nutr. 119(1): 82-88

SUBRAMANIAN MG. 1999. Alcohol inhibits suckling-induced oxytocin release in the lactating rat. Alcohol 1:51-55

SUMIDA KD, COGGER AA, MATVEYENKO AV. 2007Alcohol-induced suppression of gluconeogenesis isgreater in ethanol fed female rat hepatocytes thanmales. Alcohol 41: 67-75

TAVARES DO CARMO MG, OILER DO NASCIMENTOCM, MARTIN A, HERRERA E. 1999. Ethanol intakeduring lactation impairs milk production in rats andaffects growth and metabolism of suckling pups.Alcohol 18(1): 71-76

TAVARES DO CARMO MG, OLLER DO NASCIMENTOCM, MARTIN A, HERRERA E. 1996. Effects ofethanol intake on lipid metabolism in the lactating rat.Alcohol 13(5): 443-448

TESTAR X, LÓPEZ D, LLOBERA M, HERRERA E.1986. Ethanol administration in the drinking fluid topregnant rats as a model for the fetal alcohol síndrome.Pharmacol Biochem Behav 24(3): 625-30

TUCKER HA. 1979. Endocrinology of lactation. Semin.Perinatol. 3: 199-223

VILARÓ S, VINAS O, REMESAR X, HERRERA E. 1987.Effects of chronic ethanol consumption on lactationalperformance in rat : mammary gland and milkcomposit ion and pups’growth and metabolism.Pharmacol. Biochem. Behav. 27: 333-339

WHITWORTH NS. 1988. Lactation in humans.Psychoneuroendocrin. 13: 171-188

ZHU X; SEELIG LLJR. 2000. Developmental aspects ofintest inal intraepithelial and lamina proprialymphocytes in the rat following placental andlactat ional exposure to ethanol. Alcohol andAlcoholism 35(1): 25-30.

ethanol intake during lactation alters milk nutrient ... · azara et al. biol res 41, 2008, 317-330...

Documents