Biol. Neonate 32: 24-32 (1977)
Incorporation//? vivo of 1-,4C-Palmitic Acid into Placental and Fetal Liver Lipids of the Rabbit M.C. Elphick and D. Hull' Department of Child Health, University Hospital and Medical School, Nottingham
Key Words. Fetal rabbits • Placenta • Liver • l4C'-palmitic acid • Lipids • Incorporation Abstract. The incorporation of free fatty acid into the placental and fetal liver lipids of rabbits was studied after fetal injections of albumin-bound l-14C-palmitic acid. The fetuses were killed either 5—10 or 10-20 min after the injection. The placentas and livers were extracted for lipids and the specific activities of triglycerides (TG), phospholipids (PL), free fatty acids (FFA), monoglycerides (MG) and diglycerides (DG) measured. The lipids of the liver and placenta took up 17.0 and 3.6% of the dose, respectively, and of that the liver TG accounted for 74% and the placental TG 34% of the label in each tissue. Most of the remaining counts were in the PL fraction with the rest more or less evenly distributed between the FFA, DG and MG fractions. No activity was recorded in the cholesterol esters. The placental TG, PL, DG and MG specific activities reached the same level as that of the placental FFA, while in the liver these esters had higher specific activities (than the liver FFA). The liver TG, DG and PL had higher specific activities when compared with those of the placenta. The specific activity of the placental FFA was lower at 10-20 min than at 5—10 min; the opposite was seen for the placental TG. No time-related changes were seen in the liver lipids. It is concluded that (i) both placenta and fetal liver incorporate FFA into glycerides and PL; (ii) the liver incorporates FFA more rapidly and to a greater extent than the placenta; (iii) most of the FFA is incorporated into TG and to a lesser extent PL; (iv) in both organs hydrolysis of PL or TG occurs. These results are discussed with reference to placental transport of FFA and fetal fat metabolism.
Labelled maternal plasma free fatty acids (FFA) pass rapidly through the placenta to fetus in rats (Hummel et al., 1975); rabbits (Van Duytie et al., 1962;Elphick et al., 1975); sheep ( Van Duyne et al.. 1960); monkeys (Port-
man et al, 1969) and man (Szabo et al., 1969; Dancis et al., 1973). Measurement of umbilical venous-arterial differences in rabbits (Elphick et 1 We are grateful to the Medical Research Council for financial support and to Mrs. Janette lidson and Ms. Julie l.awlor for their skilled technical assistance.
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Introduction
al., 1975; Elphick and Hull, 1976) and man (Sabata et al., 1968; Sheath et al., 1972; El phick et al., 1976) indicate that FFA from the placenta can supply sufficient lipid for fetal adipose tissue growth. Moreover, as the mater nal FFA level increased, so also did the venousarterial difference, indicating a diffusion type of transfer o f FFA down a concentration gradi ent. It is known that the placenta rapidly esterifies fatty acids into triglycerides (TG) and phospholipids (PL), which themselves have a rapid turnover and it has been suggested that one type of placental FFA transport depends on an esterification/hydrolysis cycle in the pla cental cells (Szabo et al., 1973 b; Hummel et al., 1974b). There is also the suggestion that esterified fatty acids could be released by the pla centa into the fetal circulation (Goldwater and Stetten, 1947; Hull, 1975), in much the same way as adult liver. An examination of the incorporation of labelled palmitic acid into placental lipids and fetal liver therefore seemed pertinent to the study of placental lipid trans port and the fate of fetal circulating FFA. Tissue radioactivity was followed after fetal injections of labelled palmitic acid. The results indicate that the fetal liver is more active in esterification of fatty acids into PL and TG when compared with the placenta.
Methods Animal Procedures The rabbits, a cross between Californian and New Zealand Whites, were bred in our animal house under controlled conditions. All were studied at 28 days gestation (term = 31 days). The experimental proce dures have been described in detail elsewhere (Elphick et al.. 1975; Elphick and Hull, 1977). Briefly, the experiments were performed as follows. The pregnant doe was anaesthetized with urethane and the abdomen opened. An incision was made in the uterus and the fetal sacs were punctured, exposing the fetal neck. A
25
small cut was then made in the skin overlying the fetal external jugular vein, into which was inserted a fine polythene catheter. A dose of 12.5 pCi l-‘4C-palmitic acid in 0.25 ml albumin solution was injected through the catheter which was then flushed out with a small amount of saline. The fetus and placenta were re moved at recorded times, 5 -2 0 min after the injection and placed in cold saline. The liver was dissected from the fetus and placed in more cold saline. A second fetus was then exposed, catheterized and injected, and so on. A total of 9 injections were made into 9 fetuses from 2 does. 4 of the injected fetuses were killed 5 -1 0 min, and 5 10-20 min after the injection of labelled palmitic acid. In 1 doe the last 2 fetuses exposed were not injected, but a portion of liver and placenta were taken for analysis of radioactivity, to see if any label had been derived from previous injections into other fetuses. Chemical Methods l-14C-palmitic acid was purchased from the Radio chemical Centre, Amersham, England (specific activity > 50 /uCi/Mmol). Albumin-bound labelled fatty acids were prepared as previously described (Elphick et al., 1975). The placentas and livers were weighed and a portion (0.5-1.0 g) cut into fragments and rinsed in cold saline and extracted with 20 ml chloroformmethanol 2:1 (v/v), followed by washing with buffer at pH 6.0, as described by Folch et al. (1957). An aliquot was taken for a measure of total counts per gram tissue and another aliquot, representing about 50 mg tissue, separated into its various lipid com ponents by thin-layer chromatography (TLC) on silica gel H (Merck) in pre-equilibrated tanks containing petroleum ether 60-80 °C, diethyl ether and glacial acetic acid in the volume ratio of 60:40:1. The separated zones, after drying, were stained in iodine vapour and scraped from the plates into small test tubes. The lipids were extracted with 3 2-ml lots of chloroform-methanol 2:1 (v/v) (TG; diglycerides, DG, and monoglycerides, MG); diethyl ether-petroleum ether 40-60 °C-formic acid 50:50:1 (v/v) (fatty acids); chloroform-methanol-water-glacial acetic acid, 50:39:10:1 (v/v) (PL). The 2-ml extracts were com bined and evaporated to dryness in vacuo and taken up in 2 ml n-heptane. 1 ml of this was taken for liquid scintillation counting in 10 ml toluene-based fluid with an LKB Walac counter. Quenching was auto matically corrected for using an external standard ratio system and counting efficiency averaged 95%.
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Elphick/Hull
26
Elphick/HuU
The second half of the heptane extracts were evapo rated under air and saponified with 0.5 (V alcoholic KOH for 30 min at 65 °C followed by acidification with HC1 and extraction with chloroform-heptane, 70:30 (v/v). These extracts were suitably diluted with more chloroform-heptane, depending on the lipid zone and the fatty acid concentration measured (Elphick, 1975). It was therefore possible to estimate specific activities for tissue FFA, TG, DG (1 -3 and 1-2 combined), MG and PL. Cholesterol esters were not analysed because radioactivity was never found in this fraction.
Results
The organ weights and the total incorpora tion of label into the fetal rabbit liver and placenta are shown in table 1. Clearly, the liver contained more labelled lipids than the placenta on a unit wet weight basis and, in absolute terms, the liver took up nearly 5 times as much label as the placenta. There was no difference in the total incorporation between fetuses taken at 5 -1 0 or 10-20 min after the injection and so all these data were pooled. The relative amount of label recovered from the different lipid fractions is shown in table II.
The TG in both placenta and liver contained the highest counts, especially in the latter where the TG contained 59% of the total label incorporated into both liver and placenta com bined. This compares with a figure of 7% for the placental TG. In the placenta, the propor tion of counts, making up the total, in the FFA, DG, MG and PL fractions was compara tively greater than that in the liver. However, in absolute amounts, the liver contained more radioactivity in the DG, PL and TG fractions. The TG radioactivity in the placenta in creased from a mean of 38 X 103 dpm/g tissue Table I. Incorporation of label into fetal tissues following fetal injection of I2.5jiCi f ’C'-palmitic acid
Organ weight, g dpm/g (thousands) Incorporation of dose, %
Placenta n=9
Liver n= 7
4.86 i 0.44 210 i 30
2.38 i 0.15 2,035 ± 249
3.6 ± 0.6
17.0 ± 1.6
Numbers are means ♦ SEM of n observations.
Table 11. Total recovered label incorporated into placental and fetal liver lipids after fetal injection of 12.5 mCí 1- '■’-palmitic acid
TG FFA DG MG PL
Total radioactivity, dpm (thousands) placenta, n = 9
liver, n = 7
255 182 56 21 227
2,061 65 116 17 512
± 50 t 84 t 29 ±4 t 57
* 342 ± 12 ± 14 t 5 ± 117
Significance
p < 0.01 NS p < 0.01 NS p < 0.02
Percent of total radioactivity placenta, n = 9
liver, n = 7
34 í 4 25 ± 3 8t 1 3' 31 t 1
74 i 3 2' 4' r 18 t 3
The figures show the mean incorporation ± SEM calculated for the whole organs in n experiments. NS = Not significant. 1 SE < 0.5.
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Lipid
Incorporation of Palmitate into Placenta and Fetal Liver Lipids
27
at 5 -1 0 min to 70 X 103 dpm/g at 10—20 min. During the same time intervals, the placental FFA activity decreased from a mean of 59 X 103 dpm/g to 10 X 103 dpm/g. Such timedependent changes were not observed in any lipid fraction of the liver. The statistical signifi cance of these changes is dealt with below using data for the specific activities. Our measurements of the fatty acid content of the lipid zones on TLC plates enabled us to calculate the relative abundance of the various glycerides, PL and FFA in the two organs. The results are shown in table III from which it is evident that both organs each contain, on a unit wet weight basis, about equal quantities of PL, FFA, DG and MG. They differ in that the liver has considerably more TG. These figures should be considered as semi-quantitative only because the efficiencies of primary lipid extraction, TLC, extraction from silica gel and subsequent saponification and re-extraction have not been taken into account. For the more polar com pounds, the error may be quite large, for example for FFA recovery we have found the average efficiency of recovery to be as low as
46%. Using this correction factor for FFA, we get a tissue concentration of 5.9 ± 0.9/aEq/g, which is more than 8 times higher than the level in fetal plasma under the same experimental conditions (0.69 ± SEM 0.05 juEq/ml; Elphick e ta l, 1975). The measurements of specific activities made allowance for differences in extraction effi ciencies because the mass analysis was made on the same samples used for counting. The results are shown in table IV, which compares the pooled data for all the organs removed 5— 20 min after the injection. The different lipids in the placenta all showed equal amounts of labelling (per unit of fatty acid). The apparent ly higher specific activity of the MG was not statistically significant; the wide variation in specific activities obtained for this fraction is due to its very low tissue concentration, which made its estimation difficult, and to its low counts. The liver lipids were all more heavily labelled than those of the placenta and this was significant for TG, DG and PL. In addition, the liver lipids showed no time-related changes, although these were evident in a number of
Table III. Tissue contents of the main labelled fatty-acid-containing fractions in the lipids extracted from fetal liver and placenta
Table IV. Specific activities of the fatty acids recovered from TLC lipid zones of fetal liver and placental extracts after injection of l- '4C-palmitic acid
Lipid
Fraction dpm (thousands)AiEq of fatty acid
Fatty acid/g wet weight, jrEq placenta, n = 9
liver, n = 7
6.6 2.5 2.7 0.22 6.9
51.1 2.7 2.7 0.24 5.1
Signifi cance
placenta, n = 9 liver, n = 7 ± 1.1 ± 0.3 ± 0.5 ± 0.04 ± 0.8
t 5.7 i 0.3 t 0.8 ±0.11 t 0.8
The figures refer to the amount of fatty acids recovered after saponification of the various TLC zones and are means ± SEM.
TG FFA DG MG PL
7.9 9.8 8.8 27.6 7.8
± 2.1 ± 2.9 ± 2.2 ± 11.1 ± 2.2
21.0 10.4 32.3 59.5 46.5
± 1.4 ± 1.2 ± 9.3 ± 27.3 ± 10.5
p < 0.01 NS p < 0.02 NS p
Incorporation//? vivo of 1-,4C-Palmitic Acid into Placental and Fetal Liver Lipids of the Rabbit M.C. Elphick and D. Hull' Department of Child Health, University Hospital and Medical School, Nottingham
Key Words. Fetal rabbits • Placenta • Liver • l4C'-palmitic acid • Lipids • Incorporation Abstract. The incorporation of free fatty acid into the placental and fetal liver lipids of rabbits was studied after fetal injections of albumin-bound l-14C-palmitic acid. The fetuses were killed either 5—10 or 10-20 min after the injection. The placentas and livers were extracted for lipids and the specific activities of triglycerides (TG), phospholipids (PL), free fatty acids (FFA), monoglycerides (MG) and diglycerides (DG) measured. The lipids of the liver and placenta took up 17.0 and 3.6% of the dose, respectively, and of that the liver TG accounted for 74% and the placental TG 34% of the label in each tissue. Most of the remaining counts were in the PL fraction with the rest more or less evenly distributed between the FFA, DG and MG fractions. No activity was recorded in the cholesterol esters. The placental TG, PL, DG and MG specific activities reached the same level as that of the placental FFA, while in the liver these esters had higher specific activities (than the liver FFA). The liver TG, DG and PL had higher specific activities when compared with those of the placenta. The specific activity of the placental FFA was lower at 10-20 min than at 5—10 min; the opposite was seen for the placental TG. No time-related changes were seen in the liver lipids. It is concluded that (i) both placenta and fetal liver incorporate FFA into glycerides and PL; (ii) the liver incorporates FFA more rapidly and to a greater extent than the placenta; (iii) most of the FFA is incorporated into TG and to a lesser extent PL; (iv) in both organs hydrolysis of PL or TG occurs. These results are discussed with reference to placental transport of FFA and fetal fat metabolism.
Labelled maternal plasma free fatty acids (FFA) pass rapidly through the placenta to fetus in rats (Hummel et al., 1975); rabbits (Van Duytie et al., 1962;Elphick et al., 1975); sheep ( Van Duyne et al.. 1960); monkeys (Port-
man et al, 1969) and man (Szabo et al., 1969; Dancis et al., 1973). Measurement of umbilical venous-arterial differences in rabbits (Elphick et 1 We are grateful to the Medical Research Council for financial support and to Mrs. Janette lidson and Ms. Julie l.awlor for their skilled technical assistance.
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:06:54 AM
Introduction
al., 1975; Elphick and Hull, 1976) and man (Sabata et al., 1968; Sheath et al., 1972; El phick et al., 1976) indicate that FFA from the placenta can supply sufficient lipid for fetal adipose tissue growth. Moreover, as the mater nal FFA level increased, so also did the venousarterial difference, indicating a diffusion type of transfer o f FFA down a concentration gradi ent. It is known that the placenta rapidly esterifies fatty acids into triglycerides (TG) and phospholipids (PL), which themselves have a rapid turnover and it has been suggested that one type of placental FFA transport depends on an esterification/hydrolysis cycle in the pla cental cells (Szabo et al., 1973 b; Hummel et al., 1974b). There is also the suggestion that esterified fatty acids could be released by the pla centa into the fetal circulation (Goldwater and Stetten, 1947; Hull, 1975), in much the same way as adult liver. An examination of the incorporation of labelled palmitic acid into placental lipids and fetal liver therefore seemed pertinent to the study of placental lipid trans port and the fate of fetal circulating FFA. Tissue radioactivity was followed after fetal injections of labelled palmitic acid. The results indicate that the fetal liver is more active in esterification of fatty acids into PL and TG when compared with the placenta.
Methods Animal Procedures The rabbits, a cross between Californian and New Zealand Whites, were bred in our animal house under controlled conditions. All were studied at 28 days gestation (term = 31 days). The experimental proce dures have been described in detail elsewhere (Elphick et al.. 1975; Elphick and Hull, 1977). Briefly, the experiments were performed as follows. The pregnant doe was anaesthetized with urethane and the abdomen opened. An incision was made in the uterus and the fetal sacs were punctured, exposing the fetal neck. A
25
small cut was then made in the skin overlying the fetal external jugular vein, into which was inserted a fine polythene catheter. A dose of 12.5 pCi l-‘4C-palmitic acid in 0.25 ml albumin solution was injected through the catheter which was then flushed out with a small amount of saline. The fetus and placenta were re moved at recorded times, 5 -2 0 min after the injection and placed in cold saline. The liver was dissected from the fetus and placed in more cold saline. A second fetus was then exposed, catheterized and injected, and so on. A total of 9 injections were made into 9 fetuses from 2 does. 4 of the injected fetuses were killed 5 -1 0 min, and 5 10-20 min after the injection of labelled palmitic acid. In 1 doe the last 2 fetuses exposed were not injected, but a portion of liver and placenta were taken for analysis of radioactivity, to see if any label had been derived from previous injections into other fetuses. Chemical Methods l-14C-palmitic acid was purchased from the Radio chemical Centre, Amersham, England (specific activity > 50 /uCi/Mmol). Albumin-bound labelled fatty acids were prepared as previously described (Elphick et al., 1975). The placentas and livers were weighed and a portion (0.5-1.0 g) cut into fragments and rinsed in cold saline and extracted with 20 ml chloroformmethanol 2:1 (v/v), followed by washing with buffer at pH 6.0, as described by Folch et al. (1957). An aliquot was taken for a measure of total counts per gram tissue and another aliquot, representing about 50 mg tissue, separated into its various lipid com ponents by thin-layer chromatography (TLC) on silica gel H (Merck) in pre-equilibrated tanks containing petroleum ether 60-80 °C, diethyl ether and glacial acetic acid in the volume ratio of 60:40:1. The separated zones, after drying, were stained in iodine vapour and scraped from the plates into small test tubes. The lipids were extracted with 3 2-ml lots of chloroform-methanol 2:1 (v/v) (TG; diglycerides, DG, and monoglycerides, MG); diethyl ether-petroleum ether 40-60 °C-formic acid 50:50:1 (v/v) (fatty acids); chloroform-methanol-water-glacial acetic acid, 50:39:10:1 (v/v) (PL). The 2-ml extracts were com bined and evaporated to dryness in vacuo and taken up in 2 ml n-heptane. 1 ml of this was taken for liquid scintillation counting in 10 ml toluene-based fluid with an LKB Walac counter. Quenching was auto matically corrected for using an external standard ratio system and counting efficiency averaged 95%.
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:06:54 AM
Elphick/Hull
26
Elphick/HuU
The second half of the heptane extracts were evapo rated under air and saponified with 0.5 (V alcoholic KOH for 30 min at 65 °C followed by acidification with HC1 and extraction with chloroform-heptane, 70:30 (v/v). These extracts were suitably diluted with more chloroform-heptane, depending on the lipid zone and the fatty acid concentration measured (Elphick, 1975). It was therefore possible to estimate specific activities for tissue FFA, TG, DG (1 -3 and 1-2 combined), MG and PL. Cholesterol esters were not analysed because radioactivity was never found in this fraction.
Results
The organ weights and the total incorpora tion of label into the fetal rabbit liver and placenta are shown in table 1. Clearly, the liver contained more labelled lipids than the placenta on a unit wet weight basis and, in absolute terms, the liver took up nearly 5 times as much label as the placenta. There was no difference in the total incorporation between fetuses taken at 5 -1 0 or 10-20 min after the injection and so all these data were pooled. The relative amount of label recovered from the different lipid fractions is shown in table II.
The TG in both placenta and liver contained the highest counts, especially in the latter where the TG contained 59% of the total label incorporated into both liver and placenta com bined. This compares with a figure of 7% for the placental TG. In the placenta, the propor tion of counts, making up the total, in the FFA, DG, MG and PL fractions was compara tively greater than that in the liver. However, in absolute amounts, the liver contained more radioactivity in the DG, PL and TG fractions. The TG radioactivity in the placenta in creased from a mean of 38 X 103 dpm/g tissue Table I. Incorporation of label into fetal tissues following fetal injection of I2.5jiCi f ’C'-palmitic acid
Organ weight, g dpm/g (thousands) Incorporation of dose, %
Placenta n=9
Liver n= 7
4.86 i 0.44 210 i 30
2.38 i 0.15 2,035 ± 249
3.6 ± 0.6
17.0 ± 1.6
Numbers are means ♦ SEM of n observations.
Table 11. Total recovered label incorporated into placental and fetal liver lipids after fetal injection of 12.5 mCí 1- '■’-palmitic acid
TG FFA DG MG PL
Total radioactivity, dpm (thousands) placenta, n = 9
liver, n = 7
255 182 56 21 227
2,061 65 116 17 512
± 50 t 84 t 29 ±4 t 57
* 342 ± 12 ± 14 t 5 ± 117
Significance
p < 0.01 NS p < 0.01 NS p < 0.02
Percent of total radioactivity placenta, n = 9
liver, n = 7
34 í 4 25 ± 3 8t 1 3' 31 t 1
74 i 3 2' 4' r 18 t 3
The figures show the mean incorporation ± SEM calculated for the whole organs in n experiments. NS = Not significant. 1 SE < 0.5.
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:06:54 AM
Lipid
Incorporation of Palmitate into Placenta and Fetal Liver Lipids
27
at 5 -1 0 min to 70 X 103 dpm/g at 10—20 min. During the same time intervals, the placental FFA activity decreased from a mean of 59 X 103 dpm/g to 10 X 103 dpm/g. Such timedependent changes were not observed in any lipid fraction of the liver. The statistical signifi cance of these changes is dealt with below using data for the specific activities. Our measurements of the fatty acid content of the lipid zones on TLC plates enabled us to calculate the relative abundance of the various glycerides, PL and FFA in the two organs. The results are shown in table III from which it is evident that both organs each contain, on a unit wet weight basis, about equal quantities of PL, FFA, DG and MG. They differ in that the liver has considerably more TG. These figures should be considered as semi-quantitative only because the efficiencies of primary lipid extraction, TLC, extraction from silica gel and subsequent saponification and re-extraction have not been taken into account. For the more polar com pounds, the error may be quite large, for example for FFA recovery we have found the average efficiency of recovery to be as low as
46%. Using this correction factor for FFA, we get a tissue concentration of 5.9 ± 0.9/aEq/g, which is more than 8 times higher than the level in fetal plasma under the same experimental conditions (0.69 ± SEM 0.05 juEq/ml; Elphick e ta l, 1975). The measurements of specific activities made allowance for differences in extraction effi ciencies because the mass analysis was made on the same samples used for counting. The results are shown in table IV, which compares the pooled data for all the organs removed 5— 20 min after the injection. The different lipids in the placenta all showed equal amounts of labelling (per unit of fatty acid). The apparent ly higher specific activity of the MG was not statistically significant; the wide variation in specific activities obtained for this fraction is due to its very low tissue concentration, which made its estimation difficult, and to its low counts. The liver lipids were all more heavily labelled than those of the placenta and this was significant for TG, DG and PL. In addition, the liver lipids showed no time-related changes, although these were evident in a number of
Table III. Tissue contents of the main labelled fatty-acid-containing fractions in the lipids extracted from fetal liver and placenta
Table IV. Specific activities of the fatty acids recovered from TLC lipid zones of fetal liver and placental extracts after injection of l- '4C-palmitic acid
Lipid
Fraction dpm (thousands)AiEq of fatty acid
Fatty acid/g wet weight, jrEq placenta, n = 9
liver, n = 7
6.6 2.5 2.7 0.22 6.9
51.1 2.7 2.7 0.24 5.1
Signifi cance
placenta, n = 9 liver, n = 7 ± 1.1 ± 0.3 ± 0.5 ± 0.04 ± 0.8
t 5.7 i 0.3 t 0.8 ±0.11 t 0.8
The figures refer to the amount of fatty acids recovered after saponification of the various TLC zones and are means ± SEM.
TG FFA DG MG PL
7.9 9.8 8.8 27.6 7.8
± 2.1 ± 2.9 ± 2.2 ± 11.1 ± 2.2
21.0 10.4 32.3 59.5 46.5
± 1.4 ± 1.2 ± 9.3 ± 27.3 ± 10.5
p < 0.01 NS p < 0.02 NS p
