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Nicodne-].'-N-oxide H $(-)-Nicotine / v Nor-nicotine Nicotine glucuronide N-Methylnicotine Cotinine /-~ 3- Pyridyl--~,-methylamino)- C.arbinolamine butyraldehyde • ~,-(3-Pyridyl--z-me~hylamino)- butyric acid Nicodne ~ninium ion -y-3-Pyridyl- oxobutyric acid Cotinine is also extensively metabolized, with only about 13 to 17% excreted unchanged in ~he urine?~-~ Sever~[ rnet~bo[kes of cotinine have been reported, including trans-3"- hydroxycotinineJ~" 5"-hydroxycodnine.~ cotinine N-oxide.~ and cotinine methonium ion~ (Figure 6). Little is known abou~ ~he quantitative importance or" these metabo[ites. Hydroxycotinine appeoxs to be a major memboliteY"~s wkh urinary, concen~ons exceeding codnine by ~wo- to ~hmefo[d. Codnine N-oxide {s R minor membo[{~e {n humans, accounting Oxidative degra~don of the py~o[idinc dng fo~s "/-3-pyddy[ oxobu~dc acid nicotine an~o[ codnine, which ~s ~nher converted ~mo 3-pyddyI acetic acid (F~gum 7). compound h~ been [demificd qu~imdve[y in hum~ urine.~ Benowi[z and his co[labomzo~ have me~umd qumdtadve ~pec~ of n~codne metabolic profiles in man. L'~na~" excretion profiles of nicotine and ci~ht of i~s metaboli[cs have b~en dete~incd"~ (Figure 8). According to ~eir studies, t I) a high pc~enmgc (avenging 88%) of a systemic dose of nicotine can be accounted for by me~ufing nicotine ~d i~ metaboIites: ~2) the pattern of metabolism is similar when nicotine is inhaled or abso~ed t~nsde~alIy: t3) there is considerable intc~ndividual vadability in the pattern of metabolism, but the pattern is consistent for an individuak and (4) within individuals, the ext~nt of conjugation of nicotine ~d cotinine is highly co.elated, but neither is co.elated wi~ the extent of conjugation of Y= hyd~xycotinine. ~is suggests that similar enzymes ~ involved in the conjugation of nicodne and codnine and that a different enzyme may ~ involved in the conjugation of

5-I Cotinine-N-oxide N-Mcttaylcotinine ~ CH~ J CH~ O t Hydroxycotinine Cotinine OH Nnr-eotinine N-metlaylbutyramide ~ Cotit~e -t-3-Pyridyl- glueutoa2de oxobutyri¢ acid FIGI.~'RI~ 6. Metal0olic scheme foe, the conversion of cotinine, a major metabolite of nicotine, into hydroxy¢otinin¢, .V-me~.vleotinin,. ¢otinine N.oxide. nort:otinine, and -p.3-pyridyl oxobutyrie acid. Cotinine and its glucuronide. 3- hydroxyeotmine, its glueuronide, and ¢otinine N-oxide are d~teeted in the urine after sysmmi¢ administration of nicotine."t 3"-hydroxycotinine. 3"-Hydroxycotinine. cotinine, and nicotine account for 47%, 30%, and 15%. respectively, of the dose of nicotine in urine as free alkaloids or glucuronides. The levels of their glucuronides in urine are higher than those of their free bases. Cotinine N-oxide, nicotine N-oxide, and nomicotine are only minor metabolites in human urine and account for only 9% of the dose of nicotine. There are no studies to indicate whether this metabolic pattern remains the same or is altered during pregnancy. B. COTINI'NE PLASMA LEV'ELS: QUALITATIVE MARKER OF SMOKING The elimination half-life of nicotine after nicodne administration in man to a steady state is about 2 h?~.e'v This half-life of nicodne is useful in p~dieting its accumulation rate in the body with repetitive doses as well as the time course of its decline after cessation of dosing. .assuming a half-life of 2 h. one would predict nicotine to accumulate over 6 to 8 h (3 to 4 half- lives) of regular smoking and to persist at significant nicotine levels for 6 to 8 h after cessation of smoking. Peaks and troughs follow the use of each cigarette, but after smoking a number of cigarettes trough levels rise and the influence of peak levels becomes less important. However. a more reliable marker whose levels are maintained constant is necessary, to determine moderate use of cigarettes or other forms of tobacco. Cotinine levels are of special interest as qualitative markers and quantitative indicators of nicotine intake. Cotinine is present in the blood of smokers in much higher concentrations than

and Placental Func'tmn -~-3-Pyridyl- o×obutyric acid 3-Pyridytacetic acid FIGURE 7. Metabolic scheme t'or the conversion of .pj-pyridyl oxobutyric formed from nicotine and/or codnin+ mto .~-p.vrid¥! ~c~ic a~id (3-PAA). 3-P.~A is quafi~a~i~eiy de~ccd in urine of exl:~rnen~.al animals:' nicotine. Cotinine blood levels average about .~0 to 300 ng/ml in cigarette smokers.6~-~s After cessation of smoking, levels decline with a half-life averaging 18 to 20 h (range I I to 37 h). However, because of its tong half-tile, there is much tess fluctuation in cotinine concentrations than in nicotine concentrations throughout the day. C. PLACENTAL TRANSI~R OF NICOTINE AND ITS M~TABOLITES Three types of experimental approaches indicate that nicotine crosses the placenta: (l) securing nicotine or cotinine in umbilical blood after a mother uses tobacco preparations. (2) monitoring the effects of nicotine on the fetus, and (3) measuring placental transfer of nicotine in isolated human placental cotyledon. Nicotine has been found in amniotic fluid and umbili- cal cord blood of neonates after maternal tobacco use/~-es Nicotine infusion in pregnant sheep increases uterine vascular resistance and reduces uterine blood flow. These effects appear to be mediated by catecholamine release..°,~o Both cigarette ~moking and nicotine gum chewing increaqe fetal heart rate during the qecond mme~ter in humans, consistent with sympathetic activation.'= During the third trimester in humans, cigarette smoking or nicotine gum chewing decreases fetal heart rate and reduces fetal breathing movements, both of which may be signs of fetal hypoxia:~-7~ or nicotine- or catecholamine-induced release or" opioid peptides (enkephalins) in fetal brain:=.vs There are no detailed in ~.itro studies on the placental transfer of nicotine or its metabolites in placental cotyledon. In a preliminary, study. Sastry et al.:~ reported the transport of nicotine

i 56 Nornico~Jne Placental Toxicology ~ Nicotine 10 20 30 Percent of Go tmlne N-oxlcle Nicotine N-oxide 3-Hydroxycotinine (HC) glucuronide ' 3-HC ~Cotinine glucuronide ~ Cotinine Nicotine glucuromde 40 50 60 Systemic Dose of Nicotine in isolated perfused cotyledon of normal human term placent~ The perfusion technique is classical and. has be~n validated by several investigators.~-~ The placental cotyledon was perfused with aerated (21%. O:. 5% CO,.) IC,~bs-Ringer bicarbonate buffer (pH 7.4. 37"C~ containing 2% albumin on both the maternal (2,30 ml. 15 ml/min, 0.6 in Hg) and fetal (93 ml. 1.75 ml/min. 1.75 in Hg) sides in a closed recirculadng system. Nicotine (2 rag) was added to the maternal perfusate, and perfusate samples (I ml) were collected from both sides at regular intervals and analyzed for nicotine content by high performance liquid chromatogra- phy (HPLC~.~° In about 40 rain. tS.6q~ of the nicotine added to the maternal perfusate was transferred to the fetal pwrfusate, and the matwmal/fetal concentration redo reached 1,0: this was maintained for the next 60 rain. These results show rapid placenta[ transfer of nicotine. consistent with its high lipid solubility. D. THE HUMAN PLACENTA ~ A METABOLIC ORGAN IN SMOKERS The function of placenta as a metabolic organ is of interest in several aspects: (l) metabo- lism of nicotine and other tobacco alkaloids and components of tobacco smoke into toxic components like nitrosamines, (2) induction of placental enzymes, and (3) conversion of polyaromatic hydrocarbons (PAHs) into mutagenic metaboLites. Most of the enzymes for biowansformations present in the Liver are also present in the placenta. Induction compounds affecting liver enzymes also affect the inducible placental metabolic pathways.8t This subject has been discussed in detail in several reviews and articles.~Z-~s Glucuronidadon, sulfation, and glutathione conjugation as well as reactions dependent on cytochrome P-t-50, epoxide hydrase, catechol-O-mcthyltransferase, and monoamine oxidase

NONSS'IOKING MOTIIER villus SMOKING MO'rlIER Vaso~onszricdon of Umbilical Vcsscls Vasoconstricdo~ of Umbilica] V©sscls

6O Placental Tm-icolo~y TABLE 3 Influence of Nicotine on the Uptake of c~.Aminoisobutyric Acid ((~-A[B) by Human Placental Villi~ Treazment K~ Y,,., (n) (nmol/I) (nmoUmin/g) Irreversible component Control 114) "~ "~- ..-) _ 0.7 69 _ 8 Nicotine (8) 0.79 - 0.42 .0 _ 9 Adapted from Bamwell and Sast~') E~ch value is a mean * SE from the number (n) of placentas subjected to Ireatment with nicotine, which lowered both the Michaelis-Memen constant (K,J and maximum velo~i~ (V.m) for a-AIB 07 B. MATERNAL SMOKING AND DEPRESSION OF AMINO ACID UPTAKE BY PLACENTA The fetus is dependent on placental transfer of amino acids from maternal to fetal circu- lation for its requirements of amino acids.In The placental transfer of amino acids is a two- step process: (1) active uptake of amino acids by placental trophoblast cells from the mother's blood, and (2) passive diffusion of amino acids from placental trophoblast ceils into umbilical venous blood. The first step is critical, and its efficiency may be depressed under placental hypoxic conditions induced by maternal smoking. Nicotine and several components of to- bacco smoke (carbon monoxide, cyanides, and nitrites) reduce active uptake of amino acids by isolated human placental villi,u~.~-~-') Exposure of human placental villi to nicotine inhibits the upt~. e of ct-aminoisobutyric acid (ct-AIB). a nonmetabolizable amin6 acid. and decreases both the maximum velocity (V,,~) and the Michaelis-Menten constant (K,,) for uptake of ~-AIB'~9 (Table 3). The inhibition is neither competitive, noncompetitive, nor uncompetitive, indicating the complex nature of the inhibition of placental AIB uptake by nicodne (Figure 10). Part of the inhibition is not reversible by washing the placental tissue. which may be of significance in chronic smoking (Table 3). Concentrations of several essential amino acids (val. met. lieu. leu. tyr, pbe. his) and nonessential amino acids (asp. glu. gly. ala. arg) in the placental villi of nonsmoking mothers a,re about 30 to 50% higher than those of smokers.''--'~:~ Concentrations of thr and phe are about 14 to I5% higher in the placental villi of nonsmokers than in those of smokers. Maternal smoking decreases the uptake of amino acids by the placenta and the net transfer of amino acids from maternal to fetal blood. Therefore. fetal undernutrition for amino acids may partially explain fetal intrauterine growth retardation in tobacco smokers. C. MATERNAL SMOKING AND THE REGULATORY MECHANISMS FOR AMINO ACID UPTAKE BY PLACENTAL TROPHOBLAST CELLS c~-AIB, a nonmetabolizable amino acid. has been used as a model amino acid to study the mechanisms of amino acid uptake by placental tissue. Several regulatory mechanisms have been proposed for the cellular uptake of amino acids in the placenta: (1) the gam,maglutamyi cycle.~-'#.t~ (2) placental acetylcholine release and amino acid transport coupling.~'~-~v.~-'°.~-'o (3) phospholipid N-methylation in the plasma membraneJ~ and (4) oxidative energy sources. 12~1.129 1. The Gammaglutamyl Cycle The gammaglutamyl (GG) cycle has been postulated by Meister and Anderson~-'~ as a mechanism for amino acid transport. A hypothetical model links the GG cycle to amino acid

III i Sraoking and Pht¢'ental Function A o' ¢o " ~o ao ' ~:o ~ Nicotine (raM) 61 B ~oo! 5oo o 400 o • • 300 I00 0 -5 0 5 10 15 2o 25 1/Concentration (AIB) FIGIJ'RI~ 10. Inhibition of the uptake of ct-aminoisobu .tyric acid (a-AIB) by the human placental villus. Data ate summarized from Barnwell end Sastry.I1' (..~) Concentration*response curve for the uptake of a-aminoisobutyric acid in the presence of nicotine. "i'he ordinate represent~ the percent of control UO ratio II: inrracellular concenn'ation: O: extracellular concenmation~ for the uptake of Ct-AIB (uptake when no drug is present). No nicotine was present in the baths during preincubation. Exposure time = 2 h. Points ate means _. SE from a minimum of t0 values. The control UO ratio was 6.8 = 0.J. IB) T.vpical double reciprocal plots for the uptake of ct-AIB in the presence and absence of nicotine. Concenmation is expressed as IJJnol/l. velocity as rate of uptake in IJ.mol/g/min. The straight lines were established with linear re~,ression analysis by the method of least squares. In neither case did these lines cross on the abscissa or the ordinate. In the presence of ~ min/I. Both K~ and V,~, decreased in the presence of nicotine. (Control: V,,~ = 12 x I0"~ tool/g/rain. I~ ,, 0.26 x .10-~ tool/l: 5 rru~f, nicotine: V,,.u = 8.J, x 10-" mol/.g/min. K,, = 0.23 x 10-~ tool/l). Slopes of V,~, and K,, for ct-AIB uptake in the presence and absence of nicotine suggest that the inhibition cannot be classified as simple competitive. noncompetitive or uncomp~-.titiv¢. The results from one placenta ~re presented and -,re representative ot" eight placentas studied tsee Table I transport systems, y-Glutamyl transpeptidase tGGTP) plays a key r~le in the GG cycle. It is bound in the outside bilayer of the plasma membrane. Glutathione (glutamylcysteinylglycin~) • is continuously secreted onto the cell surt'aee, where GGTP transfers a glutamyl, group to an i incoming molecule of an amino acid. The products, glutamyl-amino acid and cysteinylglycine, are reabsorbed into the ceil. Most amino acid molecules are absorbed by independent transport systems. The number of these carrier molecules may be inversely related to the degree of

Pl¢~¢'ental Toxicology :lh,+~,.,T'.i. ;i • .i' :~;tmm;.~glul;.Ull.+, I-imfinu acid. Durra,=, ~arva~on oI" lh¢ cells I~r amino acids with low I¢veN nf extr:lccllul~lr amin+, :u'ids ~. m,,r~ mm,p~+rt cagier mtflc~ulcs arc induc+d and mco~oratcd into th¢ plasma membrane. Thu~. ~amm:~gluramyl-amino acids may s¢~'¢ as tran~mcmhran¢ or environmental signals t~r ~he uprak¢ or" amino acids by cells.~ Cells can use cbe GG cycle tbr efficient recov¢~ of cysmin¢. Human cmphoblas~ic microvilli contain high levels o~ GG~)c Maternal smokin~ du~n8 pregnancy decreases the activi=y and of GGTP in the micmvillous pl~ma mcmb~n¢~:"=~: ~T~bl¢ 4). ~is d¢cre~¢s the and abso~fion o). ~amma~luramyl-amino acids, si~nallin~ the cell m induce ~h¢ ~ynchcsis of more ~mino ~cid tran~po~ c~¢~ ~nd ~o ins~ them ~nto ~h~ plasm~ membrane. ~ere ~s an increase in ~h¢ uptake of ~-AIB. m~inly duc =o an increase in V~. in washed placenral villi from .chronic smokers (Table 4). ~e induction or" placent~[ ~-AIB tr~nspo~ ~hes durin~ m~¢emal ~mokin8 can be pa~i=lly explained by depression o[ ~e GG cycle. However, in ~be pmsenc~ of tobacco smoke component, the inc~¢d ca~¢r sysmms do nm fully compensate ~or ~be dep~ssion of the GG cycle. ~e effects of mammal smoking on componcn~ o~ ~be GG cycle ocher ~ GG~ ~¢ no~ known. 2. Placental Acetylcholine and Regulation of Amino Acid Transport The chemical transmitter function of acetylcho[ine LA.Ch) in nervous tissue is well estab- lished. Human placenta is not innervated. The evolutionary appearance of ACh preceded that of the nervous system. The ACh system is fully developed in the placenta during maturation and development of the placenta and the fetus:-~ It is synthesized in the syncytiou'ophoblast and is released into both the maternal and fetal circulations of the placenta. There are several studies which indicate a coupling link between ACh release and active uptake of amino acids from maternal blood. Atropine causes a reduction in the uptake of cc-AIB by isolated placental villous==~ or placental fragmentsJ~.~ Four different types of choline acetyltransferase (ChA) inhibitors depress the uptake of a-AIB by placental villus,n:.=:~-~-~s There is a positive corre- lation between the inhibition of ChA, ACh synthesis by ChA. and the depression of o~-AIB uph~k¢. P-xr~acellular Ca** must be present for placental release of ACh. The uptake of ce-AIB by the placental villus is depressed by 90% in Ca--free medium:-~ Inhibition of ChA decreases the synthesis of ACh and i~ release as well as a-AIB uptake in placental villi or Placentas from women with preeclampsia have higher levels of ACh than control placentas. but the outpm of ACh from preeclamptic placentas is si~mificantly reduced,tz~-*'~ The trans- port of a-AIB from the maternaJ side to the fetal side is reduced in preeclamptic peffused placenta. :'== These observations suggest tha~ feral intrauterine growth rer.~rdation in preeclamptic women can be partially explained by reduced placental ACh release and reduced amino acid transport. Clinical observations give further evidence )'or a )'unctional link between ACh and ~lacentai • ".r:.m<fer ,,f ::2:ino ;:cid.-. Hi.='h cunccnti'atioa.,, of nicotine ~gcrcase placental ACh releaseY') Maternal smoking depresses placental transfer of amino acids:~'~.0pioids decrease ACh release from placenta and depress amino acid uptake.~'-)~:-~-~ Cocaine decreases placental ACh release as well as amino acid uptake?~ Tobacco smoking and addiction to opioids or cocaine cause intrauterine growth retardation. All these observations suggest a link between ACh release and amino acid transport in placenta. There are only preliminary reports on the effects of chronic maternal smoking on the placental ACh system)~-' Maternal smoking decreases placental ChA activity (Table 4). This decrease may be due to decreased synthesis of ChA protein or inhibition of ChA. an enzyme containing -SH groups, by Cd, Ca, and Ni in tobacco smoke. Further investigations are necessary to completely evaluate the relationships among the placental AC'h system, maternal smoking, and placental amino acid transport.

Smukin:¢ and Pl~tcc, ntal t"um'ti,n TABLE 4 .Maternal Smoking and Changes in the Regulator) of Amino Acid Trunsport in Placenta" ?*lean ± .',;EM• p value t'~)r Par'amele~ Nunsmokers I.~i0 Smoker~ I,M..~ ~1~ vs. ~,1., Plasma membrane. GGTP V~ of GGTP ~nmollmg pro[c~min~ 47.39 ~ 7.~7 15.50 : 2.73 <0.o2 Pl~ memb~e. PMT PMT I ~fmol/mg p~tcin/min) 23.17 ~ I.~ 13.~) ~ 1.99 <0.02 Micmviscosi~y ~cP~ ~8 ~ 14 513 ~ 26 <O.t)2 Placental ~A ~A ,nmol ~Ch h~nncdlm~ pn~c~n/m~n 3.2 • ~-AIB upz~ ~w~shed placental viilih [~ mzio 9..SO ~ 0.4 15.30 = o.~ <0.05 K~ ~ff) 0.~ ~ 0.41 1.45 ~ 0.62 <0.05 V~, mmol/~min} 30.7 ~ ~3 • Emzh ~'aiu¢ is t~r 6-9 nonsmok~t~ or 4-.5 smokers: smukcrs u,~d 20 =,g c~garcffes/day. All va|u~s arc summarized from Sasl~" and Horst.~.~° Horsl and Sa.slryY~ Sa.slr}..,~' and Jan,,am et al.'.~ GGTP. ~ammaglutarn.~ l ~ranspepfida.,~: PMT. phospholipid N-melhyllranslcr'a.s¢: ChA. choline ac¢lyhran.~eras¢: ¢t-AIB. ¢z-aminoisobu~ync acid: I and E. in|ra- and ~xlrac~llular conccn~ratiuns of o¢-A[B: K,,..Michacli.,,-Mcmen conslan! for ~h¢ uptake of c[-AIB by placemal villi: V ..,,. maximum velocity for ~h¢ uptake of ~-A[B by isolated placental vdli. 3. Phospholipid N-Methylation in Placental Plasma Membrane and Amino Acid Transport Enzymatic methylations, in the presence of 5-adenosy[-|.-methionine [SAM) as a methyl donor, play a significant role in several cellular functions. During the past decade, three of these SAM-mediated methylations .have received special attention:'z:a'=-'~-~ (I) stepwise con- version of membrane phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine ~PME}. phosphatidyl-..VaV-dimethylethanolamine ~PMME~. and phosphatidylcholine (PC) by t~o phospholipid .\'-methyltransferases ~PMT [ and [I) in ~eve.-'al tissues: 12~ formation of protein carboxymethylesters (PCME) by protein carboxymethylase ~PCM) in several cell systems: and ~3) methylation of endogenous fatty acids by fatty acid carboxymethylases (FACM') in several tissues. These methylations in placental human trophoblast have been reviewed by Bamwell and Sastry?:: Enzymatic phospholipid N-methylation is known to occur in placental tissues. Further. it has been demonstrated that increasing the intracellular levels of 5-adenosyl-~- homocysteine (SAH), an inhibitor of phospholipid N-methylation, inhibits the uptake of c¢-AIB by the human placental villus.~= SAH is not a selective inhibitor of any specific enzymatic methylation: it inhibits all SAM-mediated enzymatic methylations. A question arises as to which one of the SAM-mediated enzymatic methylations plays a significant in the uptake ofcc-AIB by the placental villus. Amino acid uptake systems occur in the plasma m~mbrane. Barnwell and Sastry~-'v have analyzed the human placental villus for the above three groups of enzymes involved in SAM-mediated methylations.~'-~ According to these investigations, f I ) the relative distribution of PMT enzymes and amino acid carrier systems ~PCM and FACM) in the plasma membrane and placental villus homogenates and depression of (z-AIB uptake by inhibition of PMT enzymes indicate that plasma membrane phospholipid .V-methylation plays a more significant role than other types of methylations in the uptake of amino acids by the human trophoblast. tl

64 Placemal Toxicology Increasing the intracellular level of SAH blocks both phospholipid N-methylation and amino acid uptake by the placental villus under the same conditions. The blockade of amino acid transport is not complete. When phospholipid :V-methylation is blocked more than 60%. amino acid uptb, ke decreases by only about 40%. There might be a basal level of amino acid uptake which is not regulated by phospholipid N-methylation. High SAH levels inhibit PCM and FACM in addition to phospholipid N-methylation. The distribution of PMT L PMT II, PCM, and FACM as well as the amino acid carrier systems (e.g.. GGTP) indicate that phospholipid .V-methylation is possibly more intimately involved in the amino acid transport across the plasma membrane of the trophoblast than the other two enzymatic methylations. Phospholipid N-methyhransfemses contain active -SH ~roups. The cadmium ions in tobacco smoke inhibit PMTs. The PMT I activity~'~.~" of placental plasma membranes decreases in cigarette smokers~) (Table 4). However. it has yet to be evaluated whether the decrease in PMT activity, is due to a decrease in the enzyme protein or the inhibition of the enzyme by tobacco smoke components. 4. Oxidative Energy Sources and Placental Amino Acid Transport Oxygen consumption by placental slices from smoking mothers decreased in proportion to CoHb concentrations in maternal blood.~ The rate of oxygen consumption in placental slices from nonsmoking mothers ( 1.9 p,l/mgjh) is about 30% higher than that in placental slices from smoking mothers ( 1.3 gl/mg/h) at an 8% maternal CoHb concentration. These observations indicate that the energy-dependent processes of placental cells may be depressed and that the formation and concentrations of cellular ATP may be affected by hypoxia. All of the above mechanisms for the regulation of amino acid transpor~ are ATP dependent. Three ATP molecules are required for operation of the GG cycle and the uptake of one molecule of garnrnaglutamyl-amino acid. Another ATP molecule is required to form one molecule of acetyl.,coenzyrne, a substrat¢ for ChA to form one molecule of ACh. Still another ATP molecule is required to form one molecule of SAM. Three molecules of SAM are required to form one molecule of phosphatidylcholine by phospholipid methylation. Therefore. all pos- tulated regulatory, mechanisms of amino acid u'ansport in the placenta may be indirectly affected by maternal smoking. The effects of maternal smoking on placental ATP levels have ye~ to be determined. D. MATERNAL SMOKING AND TRANSFER OF AMINO ACIDS FROM THE TROPHOBLAST TO THE UMBILICAL CIRCULATION The placental trophoblasr cell membrane not only pumps amino acids into the cell but also effects a net transfer across the cells into fetal interstitial fluid and thus into fetal circulation. Most essential amino acids are actively taken up by the placenta from the maternal circulation and diffuse out into the fetal plasma. Both of these processes are influenced by maternal smoking. The former is affected by placental hypoxia and the influence of components of tobacco smoke on amino acid transpor~ systems and their regulation. The latter is influenced by both hypoxia and release of biogenic amines (catecholamines and 5-hydroxytryptamine [~- HT]) which cause constriction of.the umbilical vasculature. The rate of oxygen consumption by placental slices from smoking mothers decreases in proportion to CoHb levels in maternal blood,m Therefore, the energy-dependent processes of placental cells may b~ depressed by hypoxia. These processes include actively transporting amino acids, vitamins, and other substances. Smoking induces a certain amount of vascular damage of placental vessels. Changes in umbilical arteries, villous arterioles, and villous capillaries, have been observed in placentas from smoking mothers. These changes include a broadening of the basement membrane, increased collagen content, decreased vascularization, and pronounced intimal edema.*~:-m Depending on the degree of edema, the vascular diam- eters will decrease and hinder blood flow in the umbilical arterial-venous system.