Original Papers Biol Neonate 1990;58:61-68
© 1990 S. Karger AG, Basel 0006-3126/90/0582-0061 $2.75/0
Lactate Movements in the Term Human Placenta in situ1 François Piquarda, Adrien Schaefer a, Pierre Dellenbach b, Pascal Haberey a “Institut de Physiologie, Faculté de Médecine, Université Louis-Pasteur, Strasbourg; bCentre Médico-Chirurgical et Obstétrical de la Sécurité Sociale, Strasbourg, France
Key Words. Term human fetus • Maternal lactate concentrations • Fetal lactate concentrations • Placental lactate transfer • Placental lactate metabolism
Ruminants are the most widely used spe cies in studies of fetal nutrient requirements. Unfortunately, data derived from such ani mal experiments do not always apply to hu mans. For example, contrary to humans, lac tate in the ruminant is an important meta bolic substrate in adults as well as in the fetus. Part of the fetal lactate (LF) supply is derived from placental production [1-3]. The inability of the human fetus to use lac 1 This work was supported by grants o f the U.E.R. o f Biomedical Sciences, Strasbourg.
tate as a metabolic substrate arises from nu merous previous observations of a net flux of lactate from the fetus to the mother [4-7], However, these results were obtained in var ious acute conditions and there was no evi dence of the fetus being in a metabolic steady-state. Chronic experiments evidently cannot be performed on the human fetus, and the difficulty in obtaining valid data regarding fetal metabolism from acute stud ies has been claimed. Therefore, we have previously demonstrated, in ewes, the valid ity of the data obtained in acute experiments
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Abstract. Movements of lactate through the human placenta in situ were derived from maternal and fetal blood sampling performed under conditions that approximate as closely as possible the normal fetal metabolic state. It is reported that, at the end of pregnancy, the human fetus produces lactate which is transferred to the placenta. The actuality of this lactate transfer coupled with proton transfer is discussed taking into account the results of multiple linear regression analysis determined between the umbilical arterio-venous lactate differences and fetal and maternal lactate and proton concentrations. It is finally assumed that this lactate is partly metabolized in the placenta, the remaining part being transferred from the placenta to the mother.
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Patients and Methods This study was carried out on 77 women, who had given their consent, and their children delivered by cesarean sections. Only those elective cesarean sec tions performed for obstetric reasons (previous cesar ean section, cephalopelvic disproportion, breech pre sentation) at 39-40 weeks o f a normal pregnancy were taken into account. Any pregnancy complicated by maternal disease or fetal growth abnormality was excluded. All the cases where a fetal distress state was identified by a low birth Apgar score ( < 7) have also been excluded. During the cesarean sections, women were placed on the operating table in marked left lat eral decubitus to avoid pressure on the inferior vena cava. Only Ringer infusions were allowed. To avoid transient decreases in maternal blood pressure occur ring during conductive anesthesia [9], the women received only general anesthesia using standard and strict protocols. We have obtained consent from all patients after they had been informed o f the nature and purpose o f the investigation. Just before the incision was made in the uterine wall, blood was drawn from the iliac artery and an uterine vein. Blood was also taken from the clamped umbilical artery and vein immediately after removing the fetus and before the baby’s first breath. The blood samples, which were kept on ice until the chemical determinations were done (within the next 3 min for lactate, and 15 min for other parameters), were ana lyzed for pH, pCOi and pC>2 with a blood gas and pH analyzer (Model 168, Corning Medical, Halstead, En gland). Plasma and whole blood lactate was deter mined by means of an amperometric method (Lactate
Analyzer 640, Kontron, Basel, Switzerland) [10], Blood oxygen contents were calculated from appro priate equations and curves [11-13].
Statistics All results are presented as means ± SD. Pooled estimates o f variance were obtained using analysis o f variance, and individual differences between groups were assessed by Duncan’s multiple range test. Fetal and maternal arterio-venous (AV) differences were tested by the paired t test. Previous experiments had shown that lactate transport through mono layer [14-16] or multi layers membrane [17] took place via a specific transporter by a H+-lactate symport mechanism. Keeping in mind the working hypothesis o f a transfer o f lactate from fetus towards the placenta and mother, it was impor tant to determine the extent to which the LF uptake could be jointly dependent upon lactate and proton levels in the fetal and the maternal sides. This can be performed using the multiple linear regression analy sis. We have, therefore, determined the multiple li near regression between umbilical AV lactate differ ences (directly proportional to the fetal lactate up take) and three lots of independent variables, to an swer three queries: (l)d o the umbilical AV differ ences depend upon the maternal lactate (LM) and LF and/or proton concentrations? The four independent variables shall be the LF and proton (HF) levels, and the LM and proton (HM) levels, calculated, in each case, as the mean value o f umbilical artery and vein plasma levels or o f iliac artery and uterine vein plasma levels; (2) do the umbilical AV differences depend upon the feto-maternal gradients o f lactate and/or proton? The two independent variables shall be the differences LF - LM and HF - HM; (3) accord ing to Moll et al. [ 17], is there a facilitated diffusion o f lactate, in which lactate transfer is coupled with pro ton transfer? With such a mechanism, the net LF uptake should depend upon two opposite fluxes, a fetal one, proportional to the product o f LF and pro ton concentrations, and a maternal one, proportional to the product o f maternal levels. The two indepen dent variables shall be the products LFXHF and LMXHM. In each of the three multiple regression analysis, the multiple correlation coefficient (multiple r), its significance, and the partial correlation and the regression coefficients o f each independent variables were calculated. The significance o f the variability in the LF flux explained by the independent variables
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to study some fetal nutrients, namely elec tive cesarean sections [8]. In using this procedure, it was possible to study lactate movements through the pla centa and to determine whether the human fetus might be really a lactate ‘producer’ or not. The present study reports that at the end of pregnancy and under conditions that are as close as possible to the normal physiolog ical conditions of life in utero, the human fetus produces lactate which is transferred to the placenta.
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Lactate Fluxes in Term Human Placenta
Table 1. Biological data determined in fetal and maternal circulation during elective cesarean section Umbilical artery
Umbilical vein
Iliac artery
Uterine vein
7.270 ± 0.064a 7.313± 0.057b 7.398±0.053 pH Plasma proton 54.3 ± 8.7a 4 9 .0 ± 6 .8 b 40.3 + 4.8 nmol/1 pCO, mm Hg 50.8 ± 9.8a 41.6± 8.2» 26.6 ± 5 .9 p02 31.2 ± 8.8b 138.0 ± 26.3 19.2 ± 6.0 a mm Hg Blood lactate 1.99 ± 0.71a 1.71 ± 0 .5 Ie 1.29 ± 0.36 mmol/1 Plasma lactate mmol/1 2.25 ± 1.09a 1.96± 0.79b 1.43 ±0.51 0 2 content 5.71 ± 1.72 7.05 ± 0.82 2.93 ± 1 40a mmol/1
UmbA-UmbV IlA-UterV
7.355±0.050 - 0.043 ±0.025d 0.040 ± 0.039d 44.4 ± 5 .0
5 .3 ± 3.6d
—3.8 ± 3.8d
32.3 ± 5.5
9.3 ± 6.1d
- 5.6 ± 4.3d
56.3 ± 15.0
- 11.6 ± 6.8d
81.0 ± 25.5d
1.37 ± 0.34
0.27 ± 0.33d
-0 .0 8 ± 0 .1 7 c
1.51 ±0.52
0.29 ± 0.43d
-0 .0 9 ± 0 .2 3 c
5.46 ±1.03
- 2 .7 6 ± 1.24d
1.59 ± 0.77d
was tested by the F ratio test. Proton concentrations, expressed as nanomoles/liter, were derived from pH values. Multiple correlation analysis was made also with uterine AV lactate differences to test whether placen tal transfer of lactate from mother towards the pla centa and the fetus was conceivable.
Results The mean maternal age of the studied women was 29.4 ± 5.9 years. The fetal body weight at birth was 3,308 ± 525 grams. The time delays between the induction of anes thesia and maternal blood sampling were rather short: 6.4 ± 1.2 and 7.1 ±1. 1 min for iliac artery and uterine vein. The time
elapsed between the first maternal blood sample and the last fetal blood sample ranged from 1.53 to 4.25 min (2.31 ± 0.65 for umbilical artery and 2.43 ± 0.73 min for umbilical vein). The values of the acid-base parameters derived from venous and arterial blood are shown in table 1. Umbilical pC>2 levels are in a normal range although maternal values were high by breathing CVnitrous oxide mixture during anesthesia. Fetal lactate con centrations were higher than maternal lac tate levels (Duncan’s test, p < 0.005). The lactate AV differences show that they were positive and highly significant in fetuses (p < 0.001 for both blood and plasma lac tate). On the other hand, uterine lactate AV
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Results arc means ± SD. For pH, pCCF, pCF, plasma lactate and CF content; n - 77 for fetal values and n = 73 for maternal values. For whole blood lactate, n = 61 in fetus and mother. a p < 0.001, analysis of variance between the four vessels. b p < 0.001, cp < 0.005, fetal data (umbilical artery and vein) compared to maternal data (iliac artery and uterine vein), Duncan’s multiple range test. d p < 0.001, paired t test. c p < 0.01, paired t test.
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Table 2. The relationships between umbilical arterio-venous lactate differences (ULD) and other fetal- and maternal-independent variables as obtained through three multiple association analyses Multiple correlation coefficient
Regression coefficient
ULD depending on LF and LM and proton levels 0.224 LF 0.808 0.018 HF 0.808 -0 .0 9 2 0.808 LM - 0.001 HM 0.808
Partial correlation coefficient
0.594 0.369 0.194 0.000
FD
56 56 56 56
30.5 8.9 2.2 0.1
FSignificance
0.001 0.005 NS NS
ULD depending on LF and LM and proton differences L F -L M 0.716 0.246 0.560 H F -H M 0.716 0.016 0.319
58 58
26.5 4.1
0.001 0.05
ULD depending on lactate and proton concentration product LFXHF 0.780 0.0042 0.735 LMXHM 0.780 -0 .0 0 1 5 0.134
58 58
68.3 1.1
0.001 NS
The calculations were performed with the 61 patients in which the umbilical AV differences o f whole blood lactate, that gave the best look on the LF uptake, were available. The numbers o f freedom degree (FD) were 56, 58 and 58 for the first, the second and the third multiple correlation. FD = Freedom degrees, F = value o f the multiple analysis o f variance.
The last correlations (table 2; fig. 2), highly significant (multiple r = 0.780, F (2, 58) = 45.13; p < 0.001), establish that the fetal lac tate uptake is greatly dependent upon only the fetal product LFXHF, with a good par tial correlation coefficient. We failed to establish any significant rela tionships between uterine AV differences with either fetal lactate AV differences or those afore-mentioned independent vari ables.
Discussion The present study was undertaken in an attempt to determine the actual lactate movements through the human placenta in
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differences were negative and significant (p < 0.01). Table 2 shows the results of the three mul tiple regression analyses. A significant corre lation appears between umbilical AV lactate differences and the 4 independent variables LF, HF, LM, HM (multiple r = 0.808, F(4, 56) = 26.3; p < 0.001). The partial correla tion coefficients and their F ratio test show that only LF and HF contribute significantly in predicting the fetal lactate flux. The sec ond regressions (table 2; fig. 1) show that these AV differences are also correlated with feto-maternal differences of both lactate and proton concentrations (multiple r = 0.716, F (2, 58) = 30.47; p < 0.001), the partial cor relation coefficient indicating the more ex tensive correlation for lactate differences.
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Fig. 2. Three-dimensional re presentation o f individual values of umbilical arterio-venous lactate dif ferences (UA - UV, representing LF flux) plotted at different values of the product of LF and proton concentrations (LFXHF) and of the product of LM and proton con centrations (LMXHM). The limits o f the predicted UA - UV vs. (LFXHF) by (LMXHM) surface are calculated from the equation: UA - UV = -0 .1 0 8 8 + 0.0042 (LFXHF) - 0.0005 (LMXHM). This clearly shows that the net LF flux is only depending upon the product o f LF and proton concen trations.
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Fig. 1. Three-dimensional re presentation of individual values of umbilical arterio-venous lactate dif ferences (UA - UV, representing LF flux) plotted at different values o f feto-maternal differences o f lac tate (LF - LM) and proton (HF HM) concentrations. The limits of the predicted UA - UV vs. (LF LM) by (HF - HM) surface are cal culated from the equation: UA UV = -0 .0 2 7 + 0.246 (LF - LM) + 0.016 (HF - HM). This demon strates the additive effects o f fetomaternal lactate and proton differ ences on the net F flux.
situ at the end of gestation. The validity of the results is based on the assumption that, under acute conditions of the elective cesar ean section, fetal oxygenation and metabo lism are not markedly disturbed. It has been first reported that both LM and LF concen trations and AV lactate gradients observed in ewes, under acute conditions of cesarean section, did not differ from those obtained under chronic catheterism conditions [8], Moreover, data on the acid base balance and pC>2 in this study are in close agreement with those determined at the time of fetoscopy in the undisturbed term human fetus [18, 19]. The fetuses were not acidotic and well oxy genated during elective cesarean sections. Fi nally, it is important to note that all blood samplings were done in the 10 min following the anesthesia induction and that the time elapsed between the first maternal and the last fetal blood sample never exceeded 5 min with a mean value of 2.4 min. This time delay was not long enough to allow substan tial changes in lactate levels of both mothers and fetuses. Thus, we can state that lactate data obtained under acute conditions of this study are valuable to assess the lactate fluxes through the human term placenta in vivo. Our results, at first, demonstrate that, at the end of gestation and in steady-state con ditions, the human fetus provides lactate. This is set up on the mean positive umbilical AV differences. Such positive umbilical AV differences were already observed in more acute conditions [4, 5, 7], but also in the unstressed fetuses at the time of fetoscopy at 16-24 weeks of gestation [20, 21]. Nicolaides [22] reported that LF production should occur when the fetal oxygen content decreased below the critical level of 2 mmol/1. In this study, we have observed LF production with umbilical venous oxygen
Piquard/Schacfer/Dellenbach/Haberey
content of 5.7 mmol/1, exludingan anaerobic condition of the fetuses. Thus to our knowl edge, LF production should be considered as a norm, valid for the human species in steady-state conditions. Our data also show that LF is transferred to the placenta. The positive umbilical AV lactate differences, in addition to the fact that LF levels were always higher than LM, indicate a net transfer of lactate from the fetus to the placenta. Such a transfer is in accordance with the in vitro experiments in the human term placenta which had shown that lactate carriers existed both in the ma ternal and fetal side of the trophoblast [23] and that the lactate transfer rates were found to be the same in both maternal-tofetal and fetal-to-maternal directions at the same lactate concentrations [24], The most likely mechanism of lactate transfer through the placenta involves a facilitated diffusion of lactate, the lactate transfer being coupled with proton transfer. The results of multiple correlation analysis are consistent with such a mechanism. The more significant regres sion coefficients were obtained between the LF uptake and the LF and HF concentra tions. Moreover, the LF flux is depending not only upon the materno-fetal lactate gra dient but also upon the materno-fetal pro ton differences. That HF concentrations are always higher than IIM concentrations in creases the ability of a net transfer of lac tate from the fetus to the placenta in hu mans. The negative uterine AV differences sug gest that the fetal lactate transferred to the placenta is removed by maternal blood. In an attempt to answer the question whether the lactate supplied to the placenta by the fetus was completely or partially removed in maternal blood circulation, we have evalu
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Lactate Fluxes in Term Human Placenta
References 1 Burd LI, Jones MD, Simmons MA, et al: Placental
2
3
4
5
6
7
8
9
10
11
12
13
14
production and foetal utilization o f lactate and pyruvate: Nature 1975;254:710-711. Char VC, Creasy RK: Lactate and pyruvate as fetal metabolic substrates. Pediatr Res 1976; 10: 231-234. Comline RS, Silver M: Some aspects o f foetal and uteroplacental metabolism in cows with indwell ing umbilical and uterine vascular catheters. J Physiol 1976;260:571-586. Marx GF, Greene NM: Maternal lactate, pyru vate, and excess lactate production during labor and delivery. Am J Obstet Gynecol 1964,90:786793. Haberey P, Piquard F, Hsiung R, et al: Etude cri tique de l’acidose transmise. Rev Fr Gynécol Obstét 1981;76:877-888. Eguiluz A, Lopez Bernai A, McPherson K, et al: The use o f intrapartum fetal blood lactate mea surements for the early diagnosis o f fetal distress. Am J Obstet Gynecol 1983;147:949-954. Suidan JS, Wasserman JF, Young BK: Placental contribution to lactate production by the human fetoplacental unit. Am J Perinatol 1984; 1:306309. Piquard F, Schaefer A, Lefaivre J, et al: Acute ver sus chronic conditions for studies o f fetal nutrient requirements: Applicability to the human. Pla centa 1986;7:133-142. Morriss FH, Makowski EL, Meschia G, et al: The glucose/oxygen quotient o f the term human fetus. Biol Neonate 1975;25:44-52. Piquard F, Schaefer A, Haberey P, et al: Rapid bedside estimation o f plasma and whole blood lac tic acid. Intensive Care Med 1980;7:35-38. Beer R, Bardels H, Raczkowski HP: Die Sauerstoffdissoziationskurve des fetalen Blutes und der Gasaustausch in der menschlichen Placenta. Pfliigers Arch 1955;260:306-319. Kelman GR, Nunn JF: Nomograms for correction o f blood p 0 2, p C 0 2, pH and base excess for time and temperature. J Appl Physiol 1966;21:1484— 1490. Kirschbaum TH, DeHaven JC, Shapiro N, et al: Oxyhemoglobin dissociation characteristics o f hu man and sheep maternal and fetal blood. Am J Obstet Gynecol 1966;96:741-759. Dubinsky WP, Racker E: The mechanism o f lac-
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ated the uterine/umbilical blood flow ratio from O 2 contents on both sides of the placen ta. Taking into account that about 17% of maternal oxygen supplied to the conceptus is diverted to support placental metabolism [25], it can be stated that uterine and umbil ical AV O 2 content differences are due only to differences in fetal and maternal blood flows. We have used the Fick’s law to deter mine the ratio of blood flows: Rbf = (AV fetal O 2 content)/(AV maternal O 2 content X0.83). A mean ratio of 2.11 ± 1.28 was thus obtained. The level of this ratio is vali dated by the reported values of a mean um bilical blood flow of 362 ml/min [26, 27] and a mean uterine blood flow of 705 ml/min [26, 28], giving an uterine/umbilical blood flow ratio of 1.95. From the ratio value and using whole blood lactate AV differences we have calculated the fraction of the lactate transferred to the mother (AV maternal blood lactate X 2.11). We have found that, for every 0.27 ± 0.33 mmol of whole blood lactate/liter of blood supplied by the fetus to the placenta, 0.18 ± 0.29 mmol/1 was trans ferred to the mother. The remaining part (0.094 ± 0.250 mmol/1) was metabolized by the placenta (p < 0.01, paired t test). Thus, approximately one third of the lactate transfered by the fetus is metabolized by the pla centa. In this regard, our results do not agree with a placental production of lactate in vitro [29, 30] which, however, may be due to a ‘wash-out’ effect during perfusion [30, 31]. The placental lactate metabolism could be related to that of alanine (via the pyruvate) in so far as the placenta produces alanine in normal conditions [32, 33] and an alanine aminotransferase activity was found in the cytosolic fraction of human placenta [34]. Further studies on this hypothesis should be done.
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20
21
22
23
24
25
tate transport in human erythrocytes. J Membr Biol 1978;44:25-36. De Bruijne AW, Vreeburg H, Van Stevenink J: Kinetic analysis o f L-Lactate transport in human erythrocytes via the monocarboxylate-specific carrier system. Biochim Biophys Acta 1983;732: 562-568. Monson JP, Smith JA, Cohen RD, et al: Evidence for a lactate transporter in the plasma membrane o f the rat hepatocyte. Clin Sci 1982;62:411-420. Moll W, Girard H, Gros G: Facilitated diffusion o f lactic acid in the guinea-pig placenta. Pfliigers Arch 1980;385:229-238. Mackenzie IZ, Castle B, Johnson P: Blood gas analysis in the unstressed human fetus at 17-22 week’s gestation; in Rolfe P (ed): Fetal Physiolog ical Measurements. London, Butterworths, 1986, pp 156-163. Bozzetti P, Buscaglia M, Cetin I, et al: Respiratory gases, acid base balance and lactate concentra tions of the midterm human fetus. Biol Neonate 1987;51:188-197. Aynsley-Green A, Soltesz G, Jenkins PA, et al: The metabolic and endocrine milieu o f the human fetus at 18-21 weeks o f gestation. II. Blood glu cose, lactate, pyruvate and ketone body concen trations. Biol Neonate 1985;47:19-25. Soothill PW, Nicolaides KH, Rodeck CH, et al: Blood gases and acid-base status o f the human sec ond trimester foetus. Obstet. Gynecol 1986;68: 173-176. Nicolaides KH: Studies on fetal physiology and pathophysiology in rhesus disease. Semin Perina tal 1989;13:328-339. Carstensen MH, Leichtweiss HP, Schroder H: Lactate carriers in the artificially perfused human term placenta. Placenta 1983;4:165-174. Ulsley NP, Wootton R, Penfold P, et al: Lactate transfer across the perfused human placenta. Pla centa 1986;7:209-220. Challier JC, Schneider H, Dancis J: In vitro perfu sion of human placenta V. oxygen consumption. Am J Obstet Gynecol 1976;126:261-265.
26 Faber JJ, Thornburg KL: Placental blood flows; in Faber JJ, Thornburg KL (eds): Placental Physiolo gy. New York, Raven Press, 1983, pp 41-46. 27 Van Lierde M, Oberweiss D, Thomas K: Ultra sonic measurement o f aortic and umbilical blood flow in the human fetus. Obstet Gynecol 1984;63: 801-805. 28 Assali NS, Douglas RA, Baird WW; et al: Mea surement o f uterine blood flow and uterine metab olism. Am J Obstet Gynecol 1953;66:248-253. 29 Holzman IR, Philipps AS, Battaglia FC: Glucose metabolism, lactate and ammonia production by the human placenta in vitro, Pediatr Res 1979; 13: 117-120. 30 Hauguel S, Challier JC, Cedard L, et al: Metabo lism of the human placenta perfused in vitro: Glu cose transfer and utilization, O 2 consumption, lac tate and ammonia production. Pediatr Res 1983; 17:729-732. 31 Carroll MJ: The influence o f perfusion in situ on lactate and pyruvate levels in guinea pig placenta. Placenta 1981;2:271-274. 32 Prenton MA, Young M: Umbilical vein-artery and uterine arteriovenous plasma amino acid dif ferences in human subjects. J Obstet Gynaecol Br Cwlth 1969;76:404-411. 33 Schaefer A, Ahn HS, Nisand I, et al: Echanges transplacentaires des acides aminés en fin de ges tation chez le sujet humain. J Physiol (Paris) 1979;75:67A. 34 Matalon R, Michals K: Gluconeogenic enzymes in the human placenta. J Inherited Metab Dis 1984; 7:179-181.
François Piquard Institut de Physiologie Faculté de Médecine Université Louis-Pasteur F-67085 Strasbourg Cedex (France)
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Piquard/Schaefer/Dellenbach/Haberey
© 1990 S. Karger AG, Basel 0006-3126/90/0582-0061 $2.75/0
Lactate Movements in the Term Human Placenta in situ1 François Piquarda, Adrien Schaefer a, Pierre Dellenbach b, Pascal Haberey a “Institut de Physiologie, Faculté de Médecine, Université Louis-Pasteur, Strasbourg; bCentre Médico-Chirurgical et Obstétrical de la Sécurité Sociale, Strasbourg, France
Key Words. Term human fetus • Maternal lactate concentrations • Fetal lactate concentrations • Placental lactate transfer • Placental lactate metabolism
Ruminants are the most widely used spe cies in studies of fetal nutrient requirements. Unfortunately, data derived from such ani mal experiments do not always apply to hu mans. For example, contrary to humans, lac tate in the ruminant is an important meta bolic substrate in adults as well as in the fetus. Part of the fetal lactate (LF) supply is derived from placental production [1-3]. The inability of the human fetus to use lac 1 This work was supported by grants o f the U.E.R. o f Biomedical Sciences, Strasbourg.
tate as a metabolic substrate arises from nu merous previous observations of a net flux of lactate from the fetus to the mother [4-7], However, these results were obtained in var ious acute conditions and there was no evi dence of the fetus being in a metabolic steady-state. Chronic experiments evidently cannot be performed on the human fetus, and the difficulty in obtaining valid data regarding fetal metabolism from acute stud ies has been claimed. Therefore, we have previously demonstrated, in ewes, the valid ity of the data obtained in acute experiments
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/22/2018 9:36:02 PM
Abstract. Movements of lactate through the human placenta in situ were derived from maternal and fetal blood sampling performed under conditions that approximate as closely as possible the normal fetal metabolic state. It is reported that, at the end of pregnancy, the human fetus produces lactate which is transferred to the placenta. The actuality of this lactate transfer coupled with proton transfer is discussed taking into account the results of multiple linear regression analysis determined between the umbilical arterio-venous lactate differences and fetal and maternal lactate and proton concentrations. It is finally assumed that this lactate is partly metabolized in the placenta, the remaining part being transferred from the placenta to the mother.
Piquard/Schaefer/Dellenbach/Haberey
62
Patients and Methods This study was carried out on 77 women, who had given their consent, and their children delivered by cesarean sections. Only those elective cesarean sec tions performed for obstetric reasons (previous cesar ean section, cephalopelvic disproportion, breech pre sentation) at 39-40 weeks o f a normal pregnancy were taken into account. Any pregnancy complicated by maternal disease or fetal growth abnormality was excluded. All the cases where a fetal distress state was identified by a low birth Apgar score ( < 7) have also been excluded. During the cesarean sections, women were placed on the operating table in marked left lat eral decubitus to avoid pressure on the inferior vena cava. Only Ringer infusions were allowed. To avoid transient decreases in maternal blood pressure occur ring during conductive anesthesia [9], the women received only general anesthesia using standard and strict protocols. We have obtained consent from all patients after they had been informed o f the nature and purpose o f the investigation. Just before the incision was made in the uterine wall, blood was drawn from the iliac artery and an uterine vein. Blood was also taken from the clamped umbilical artery and vein immediately after removing the fetus and before the baby’s first breath. The blood samples, which were kept on ice until the chemical determinations were done (within the next 3 min for lactate, and 15 min for other parameters), were ana lyzed for pH, pCOi and pC>2 with a blood gas and pH analyzer (Model 168, Corning Medical, Halstead, En gland). Plasma and whole blood lactate was deter mined by means of an amperometric method (Lactate
Analyzer 640, Kontron, Basel, Switzerland) [10], Blood oxygen contents were calculated from appro priate equations and curves [11-13].
Statistics All results are presented as means ± SD. Pooled estimates o f variance were obtained using analysis o f variance, and individual differences between groups were assessed by Duncan’s multiple range test. Fetal and maternal arterio-venous (AV) differences were tested by the paired t test. Previous experiments had shown that lactate transport through mono layer [14-16] or multi layers membrane [17] took place via a specific transporter by a H+-lactate symport mechanism. Keeping in mind the working hypothesis o f a transfer o f lactate from fetus towards the placenta and mother, it was impor tant to determine the extent to which the LF uptake could be jointly dependent upon lactate and proton levels in the fetal and the maternal sides. This can be performed using the multiple linear regression analy sis. We have, therefore, determined the multiple li near regression between umbilical AV lactate differ ences (directly proportional to the fetal lactate up take) and three lots of independent variables, to an swer three queries: (l)d o the umbilical AV differ ences depend upon the maternal lactate (LM) and LF and/or proton concentrations? The four independent variables shall be the LF and proton (HF) levels, and the LM and proton (HM) levels, calculated, in each case, as the mean value o f umbilical artery and vein plasma levels or o f iliac artery and uterine vein plasma levels; (2) do the umbilical AV differences depend upon the feto-maternal gradients o f lactate and/or proton? The two independent variables shall be the differences LF - LM and HF - HM; (3) accord ing to Moll et al. [ 17], is there a facilitated diffusion o f lactate, in which lactate transfer is coupled with pro ton transfer? With such a mechanism, the net LF uptake should depend upon two opposite fluxes, a fetal one, proportional to the product o f LF and pro ton concentrations, and a maternal one, proportional to the product o f maternal levels. The two indepen dent variables shall be the products LFXHF and LMXHM. In each of the three multiple regression analysis, the multiple correlation coefficient (multiple r), its significance, and the partial correlation and the regression coefficients o f each independent variables were calculated. The significance o f the variability in the LF flux explained by the independent variables
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 6/22/2018 9:36:02 PM
to study some fetal nutrients, namely elec tive cesarean sections [8]. In using this procedure, it was possible to study lactate movements through the pla centa and to determine whether the human fetus might be really a lactate ‘producer’ or not. The present study reports that at the end of pregnancy and under conditions that are as close as possible to the normal physiolog ical conditions of life in utero, the human fetus produces lactate which is transferred to the placenta.
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Lactate Fluxes in Term Human Placenta
Table 1. Biological data determined in fetal and maternal circulation during elective cesarean section Umbilical artery
Umbilical vein
Iliac artery
Uterine vein
7.270 ± 0.064a 7.313± 0.057b 7.398±0.053 pH Plasma proton 54.3 ± 8.7a 4 9 .0 ± 6 .8 b 40.3 + 4.8 nmol/1 pCO, mm Hg 50.8 ± 9.8a 41.6± 8.2» 26.6 ± 5 .9 p02 31.2 ± 8.8b 138.0 ± 26.3 19.2 ± 6.0 a mm Hg Blood lactate 1.99 ± 0.71a 1.71 ± 0 .5 Ie 1.29 ± 0.36 mmol/1 Plasma lactate mmol/1 2.25 ± 1.09a 1.96± 0.79b 1.43 ±0.51 0 2 content 5.71 ± 1.72 7.05 ± 0.82 2.93 ± 1 40a mmol/1
UmbA-UmbV IlA-UterV
7.355±0.050 - 0.043 ±0.025d 0.040 ± 0.039d 44.4 ± 5 .0
5 .3 ± 3.6d
—3.8 ± 3.8d
32.3 ± 5.5
9.3 ± 6.1d
- 5.6 ± 4.3d
56.3 ± 15.0
- 11.6 ± 6.8d
81.0 ± 25.5d
1.37 ± 0.34
0.27 ± 0.33d
-0 .0 8 ± 0 .1 7 c
1.51 ±0.52
0.29 ± 0.43d
-0 .0 9 ± 0 .2 3 c
5.46 ±1.03
- 2 .7 6 ± 1.24d
1.59 ± 0.77d
was tested by the F ratio test. Proton concentrations, expressed as nanomoles/liter, were derived from pH values. Multiple correlation analysis was made also with uterine AV lactate differences to test whether placen tal transfer of lactate from mother towards the pla centa and the fetus was conceivable.
Results The mean maternal age of the studied women was 29.4 ± 5.9 years. The fetal body weight at birth was 3,308 ± 525 grams. The time delays between the induction of anes thesia and maternal blood sampling were rather short: 6.4 ± 1.2 and 7.1 ±1. 1 min for iliac artery and uterine vein. The time
elapsed between the first maternal blood sample and the last fetal blood sample ranged from 1.53 to 4.25 min (2.31 ± 0.65 for umbilical artery and 2.43 ± 0.73 min for umbilical vein). The values of the acid-base parameters derived from venous and arterial blood are shown in table 1. Umbilical pC>2 levels are in a normal range although maternal values were high by breathing CVnitrous oxide mixture during anesthesia. Fetal lactate con centrations were higher than maternal lac tate levels (Duncan’s test, p < 0.005). The lactate AV differences show that they were positive and highly significant in fetuses (p < 0.001 for both blood and plasma lac tate). On the other hand, uterine lactate AV
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Results arc means ± SD. For pH, pCCF, pCF, plasma lactate and CF content; n - 77 for fetal values and n = 73 for maternal values. For whole blood lactate, n = 61 in fetus and mother. a p < 0.001, analysis of variance between the four vessels. b p < 0.001, cp < 0.005, fetal data (umbilical artery and vein) compared to maternal data (iliac artery and uterine vein), Duncan’s multiple range test. d p < 0.001, paired t test. c p < 0.01, paired t test.
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Table 2. The relationships between umbilical arterio-venous lactate differences (ULD) and other fetal- and maternal-independent variables as obtained through three multiple association analyses Multiple correlation coefficient
Regression coefficient
ULD depending on LF and LM and proton levels 0.224 LF 0.808 0.018 HF 0.808 -0 .0 9 2 0.808 LM - 0.001 HM 0.808
Partial correlation coefficient
0.594 0.369 0.194 0.000
FD
56 56 56 56
30.5 8.9 2.2 0.1
FSignificance
0.001 0.005 NS NS
ULD depending on LF and LM and proton differences L F -L M 0.716 0.246 0.560 H F -H M 0.716 0.016 0.319
58 58
26.5 4.1
0.001 0.05
ULD depending on lactate and proton concentration product LFXHF 0.780 0.0042 0.735 LMXHM 0.780 -0 .0 0 1 5 0.134
58 58
68.3 1.1
0.001 NS
The calculations were performed with the 61 patients in which the umbilical AV differences o f whole blood lactate, that gave the best look on the LF uptake, were available. The numbers o f freedom degree (FD) were 56, 58 and 58 for the first, the second and the third multiple correlation. FD = Freedom degrees, F = value o f the multiple analysis o f variance.
The last correlations (table 2; fig. 2), highly significant (multiple r = 0.780, F (2, 58) = 45.13; p < 0.001), establish that the fetal lac tate uptake is greatly dependent upon only the fetal product LFXHF, with a good par tial correlation coefficient. We failed to establish any significant rela tionships between uterine AV differences with either fetal lactate AV differences or those afore-mentioned independent vari ables.
Discussion The present study was undertaken in an attempt to determine the actual lactate movements through the human placenta in
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differences were negative and significant (p < 0.01). Table 2 shows the results of the three mul tiple regression analyses. A significant corre lation appears between umbilical AV lactate differences and the 4 independent variables LF, HF, LM, HM (multiple r = 0.808, F(4, 56) = 26.3; p < 0.001). The partial correla tion coefficients and their F ratio test show that only LF and HF contribute significantly in predicting the fetal lactate flux. The sec ond regressions (table 2; fig. 1) show that these AV differences are also correlated with feto-maternal differences of both lactate and proton concentrations (multiple r = 0.716, F (2, 58) = 30.47; p < 0.001), the partial cor relation coefficient indicating the more ex tensive correlation for lactate differences.
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Fig. 2. Three-dimensional re presentation o f individual values of umbilical arterio-venous lactate dif ferences (UA - UV, representing LF flux) plotted at different values of the product of LF and proton concentrations (LFXHF) and of the product of LM and proton con centrations (LMXHM). The limits o f the predicted UA - UV vs. (LFXHF) by (LMXHM) surface are calculated from the equation: UA - UV = -0 .1 0 8 8 + 0.0042 (LFXHF) - 0.0005 (LMXHM). This clearly shows that the net LF flux is only depending upon the product o f LF and proton concen trations.
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Fig. 1. Three-dimensional re presentation of individual values of umbilical arterio-venous lactate dif ferences (UA - UV, representing LF flux) plotted at different values o f feto-maternal differences o f lac tate (LF - LM) and proton (HF HM) concentrations. The limits of the predicted UA - UV vs. (LF LM) by (HF - HM) surface are cal culated from the equation: UA UV = -0 .0 2 7 + 0.246 (LF - LM) + 0.016 (HF - HM). This demon strates the additive effects o f fetomaternal lactate and proton differ ences on the net F flux.
situ at the end of gestation. The validity of the results is based on the assumption that, under acute conditions of the elective cesar ean section, fetal oxygenation and metabo lism are not markedly disturbed. It has been first reported that both LM and LF concen trations and AV lactate gradients observed in ewes, under acute conditions of cesarean section, did not differ from those obtained under chronic catheterism conditions [8], Moreover, data on the acid base balance and pC>2 in this study are in close agreement with those determined at the time of fetoscopy in the undisturbed term human fetus [18, 19]. The fetuses were not acidotic and well oxy genated during elective cesarean sections. Fi nally, it is important to note that all blood samplings were done in the 10 min following the anesthesia induction and that the time elapsed between the first maternal and the last fetal blood sample never exceeded 5 min with a mean value of 2.4 min. This time delay was not long enough to allow substan tial changes in lactate levels of both mothers and fetuses. Thus, we can state that lactate data obtained under acute conditions of this study are valuable to assess the lactate fluxes through the human term placenta in vivo. Our results, at first, demonstrate that, at the end of gestation and in steady-state con ditions, the human fetus provides lactate. This is set up on the mean positive umbilical AV differences. Such positive umbilical AV differences were already observed in more acute conditions [4, 5, 7], but also in the unstressed fetuses at the time of fetoscopy at 16-24 weeks of gestation [20, 21]. Nicolaides [22] reported that LF production should occur when the fetal oxygen content decreased below the critical level of 2 mmol/1. In this study, we have observed LF production with umbilical venous oxygen
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content of 5.7 mmol/1, exludingan anaerobic condition of the fetuses. Thus to our knowl edge, LF production should be considered as a norm, valid for the human species in steady-state conditions. Our data also show that LF is transferred to the placenta. The positive umbilical AV lactate differences, in addition to the fact that LF levels were always higher than LM, indicate a net transfer of lactate from the fetus to the placenta. Such a transfer is in accordance with the in vitro experiments in the human term placenta which had shown that lactate carriers existed both in the ma ternal and fetal side of the trophoblast [23] and that the lactate transfer rates were found to be the same in both maternal-tofetal and fetal-to-maternal directions at the same lactate concentrations [24], The most likely mechanism of lactate transfer through the placenta involves a facilitated diffusion of lactate, the lactate transfer being coupled with proton transfer. The results of multiple correlation analysis are consistent with such a mechanism. The more significant regres sion coefficients were obtained between the LF uptake and the LF and HF concentra tions. Moreover, the LF flux is depending not only upon the materno-fetal lactate gra dient but also upon the materno-fetal pro ton differences. That HF concentrations are always higher than IIM concentrations in creases the ability of a net transfer of lac tate from the fetus to the placenta in hu mans. The negative uterine AV differences sug gest that the fetal lactate transferred to the placenta is removed by maternal blood. In an attempt to answer the question whether the lactate supplied to the placenta by the fetus was completely or partially removed in maternal blood circulation, we have evalu
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Lactate Fluxes in Term Human Placenta
References 1 Burd LI, Jones MD, Simmons MA, et al: Placental
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production and foetal utilization o f lactate and pyruvate: Nature 1975;254:710-711. Char VC, Creasy RK: Lactate and pyruvate as fetal metabolic substrates. Pediatr Res 1976; 10: 231-234. Comline RS, Silver M: Some aspects o f foetal and uteroplacental metabolism in cows with indwell ing umbilical and uterine vascular catheters. J Physiol 1976;260:571-586. Marx GF, Greene NM: Maternal lactate, pyru vate, and excess lactate production during labor and delivery. Am J Obstet Gynecol 1964,90:786793. Haberey P, Piquard F, Hsiung R, et al: Etude cri tique de l’acidose transmise. Rev Fr Gynécol Obstét 1981;76:877-888. Eguiluz A, Lopez Bernai A, McPherson K, et al: The use o f intrapartum fetal blood lactate mea surements for the early diagnosis o f fetal distress. Am J Obstet Gynecol 1983;147:949-954. Suidan JS, Wasserman JF, Young BK: Placental contribution to lactate production by the human fetoplacental unit. Am J Perinatol 1984; 1:306309. Piquard F, Schaefer A, Lefaivre J, et al: Acute ver sus chronic conditions for studies o f fetal nutrient requirements: Applicability to the human. Pla centa 1986;7:133-142. Morriss FH, Makowski EL, Meschia G, et al: The glucose/oxygen quotient o f the term human fetus. Biol Neonate 1975;25:44-52. Piquard F, Schaefer A, Haberey P, et al: Rapid bedside estimation o f plasma and whole blood lac tic acid. Intensive Care Med 1980;7:35-38. Beer R, Bardels H, Raczkowski HP: Die Sauerstoffdissoziationskurve des fetalen Blutes und der Gasaustausch in der menschlichen Placenta. Pfliigers Arch 1955;260:306-319. Kelman GR, Nunn JF: Nomograms for correction o f blood p 0 2, p C 0 2, pH and base excess for time and temperature. J Appl Physiol 1966;21:1484— 1490. Kirschbaum TH, DeHaven JC, Shapiro N, et al: Oxyhemoglobin dissociation characteristics o f hu man and sheep maternal and fetal blood. Am J Obstet Gynecol 1966;96:741-759. Dubinsky WP, Racker E: The mechanism o f lac-
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ated the uterine/umbilical blood flow ratio from O 2 contents on both sides of the placen ta. Taking into account that about 17% of maternal oxygen supplied to the conceptus is diverted to support placental metabolism [25], it can be stated that uterine and umbil ical AV O 2 content differences are due only to differences in fetal and maternal blood flows. We have used the Fick’s law to deter mine the ratio of blood flows: Rbf = (AV fetal O 2 content)/(AV maternal O 2 content X0.83). A mean ratio of 2.11 ± 1.28 was thus obtained. The level of this ratio is vali dated by the reported values of a mean um bilical blood flow of 362 ml/min [26, 27] and a mean uterine blood flow of 705 ml/min [26, 28], giving an uterine/umbilical blood flow ratio of 1.95. From the ratio value and using whole blood lactate AV differences we have calculated the fraction of the lactate transferred to the mother (AV maternal blood lactate X 2.11). We have found that, for every 0.27 ± 0.33 mmol of whole blood lactate/liter of blood supplied by the fetus to the placenta, 0.18 ± 0.29 mmol/1 was trans ferred to the mother. The remaining part (0.094 ± 0.250 mmol/1) was metabolized by the placenta (p < 0.01, paired t test). Thus, approximately one third of the lactate transfered by the fetus is metabolized by the pla centa. In this regard, our results do not agree with a placental production of lactate in vitro [29, 30] which, however, may be due to a ‘wash-out’ effect during perfusion [30, 31]. The placental lactate metabolism could be related to that of alanine (via the pyruvate) in so far as the placenta produces alanine in normal conditions [32, 33] and an alanine aminotransferase activity was found in the cytosolic fraction of human placenta [34]. Further studies on this hypothesis should be done.
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tate transport in human erythrocytes. J Membr Biol 1978;44:25-36. De Bruijne AW, Vreeburg H, Van Stevenink J: Kinetic analysis o f L-Lactate transport in human erythrocytes via the monocarboxylate-specific carrier system. Biochim Biophys Acta 1983;732: 562-568. Monson JP, Smith JA, Cohen RD, et al: Evidence for a lactate transporter in the plasma membrane o f the rat hepatocyte. Clin Sci 1982;62:411-420. Moll W, Girard H, Gros G: Facilitated diffusion o f lactic acid in the guinea-pig placenta. Pfliigers Arch 1980;385:229-238. Mackenzie IZ, Castle B, Johnson P: Blood gas analysis in the unstressed human fetus at 17-22 week’s gestation; in Rolfe P (ed): Fetal Physiolog ical Measurements. London, Butterworths, 1986, pp 156-163. Bozzetti P, Buscaglia M, Cetin I, et al: Respiratory gases, acid base balance and lactate concentra tions of the midterm human fetus. Biol Neonate 1987;51:188-197. Aynsley-Green A, Soltesz G, Jenkins PA, et al: The metabolic and endocrine milieu o f the human fetus at 18-21 weeks o f gestation. II. Blood glu cose, lactate, pyruvate and ketone body concen trations. Biol Neonate 1985;47:19-25. Soothill PW, Nicolaides KH, Rodeck CH, et al: Blood gases and acid-base status o f the human sec ond trimester foetus. Obstet. Gynecol 1986;68: 173-176. Nicolaides KH: Studies on fetal physiology and pathophysiology in rhesus disease. Semin Perina tal 1989;13:328-339. Carstensen MH, Leichtweiss HP, Schroder H: Lactate carriers in the artificially perfused human term placenta. Placenta 1983;4:165-174. Ulsley NP, Wootton R, Penfold P, et al: Lactate transfer across the perfused human placenta. Pla centa 1986;7:209-220. Challier JC, Schneider H, Dancis J: In vitro perfu sion of human placenta V. oxygen consumption. Am J Obstet Gynecol 1976;126:261-265.
26 Faber JJ, Thornburg KL: Placental blood flows; in Faber JJ, Thornburg KL (eds): Placental Physiolo gy. New York, Raven Press, 1983, pp 41-46. 27 Van Lierde M, Oberweiss D, Thomas K: Ultra sonic measurement o f aortic and umbilical blood flow in the human fetus. Obstet Gynecol 1984;63: 801-805. 28 Assali NS, Douglas RA, Baird WW; et al: Mea surement o f uterine blood flow and uterine metab olism. Am J Obstet Gynecol 1953;66:248-253. 29 Holzman IR, Philipps AS, Battaglia FC: Glucose metabolism, lactate and ammonia production by the human placenta in vitro, Pediatr Res 1979; 13: 117-120. 30 Hauguel S, Challier JC, Cedard L, et al: Metabo lism of the human placenta perfused in vitro: Glu cose transfer and utilization, O 2 consumption, lac tate and ammonia production. Pediatr Res 1983; 17:729-732. 31 Carroll MJ: The influence o f perfusion in situ on lactate and pyruvate levels in guinea pig placenta. Placenta 1981;2:271-274. 32 Prenton MA, Young M: Umbilical vein-artery and uterine arteriovenous plasma amino acid dif ferences in human subjects. J Obstet Gynaecol Br Cwlth 1969;76:404-411. 33 Schaefer A, Ahn HS, Nisand I, et al: Echanges transplacentaires des acides aminés en fin de ges tation chez le sujet humain. J Physiol (Paris) 1979;75:67A. 34 Matalon R, Michals K: Gluconeogenic enzymes in the human placenta. J Inherited Metab Dis 1984; 7:179-181.
François Piquard Institut de Physiologie Faculté de Médecine Université Louis-Pasteur F-67085 Strasbourg Cedex (France)
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