Effect of ischemia on fatty acid metabolism in fetal lung

Effect of ischemia on fatty acid metabolism in fetal lung

Life Sciences, Vol. 33, pp. 569-576 Printed in the U.S.A. Pergamon Press EFFECT OF ISCHEMIA ON FATTY ACID METABOLISM IN FETAL LUNG Dipak K. Das, Jah...

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Life Sciences, Vol. 33, pp. 569-576 Printed in the U.S.A.

Pergamon Press

EFFECT OF ISCHEMIA ON FATTY ACID METABOLISM IN FETAL LUNG Dipak K. Das, Jahangir Ayromlooi and Anita Neogi Division of Pulmonary Medicine and Department of Obstetrics Long Island Jewish-Hillside Medical Center New Hyde Park, New York 11042

and Gynecology

(Received in final form May 24, 1983) Summary The effects of ischemia on in vivo fatty acid metabolism in fetal lung were studied using rabbit fetuses of 25 to 28 gestational age. Ischemia was produced by inflating the aortic balloon thereby reducing the uterine blood flow. Ischemic insult resulted significant increase in lactate/pyruvate and NADH/NAD ratios and decrease in ATP/ADP ratio in fetal lung. Levels of CoA, acetyl CoA, carnitine and acetyl carnitine decreased while those of long chain acyl CoA and long chain acyl carnitine enhanced. Tissue content of these metabolites returned to normal after 2 hr stabilization following 20 min of ischemic insult. Ischemia also caused small increase in lipogenesis and neutral lipid content of fetal lungs. Our results thus suggest t h a t , - o x i d a tion in fetal lung is inhibited and becomes rate-limiting for fatty acid oxidation during ischemia. Sudden occurrence of hypoxia or ischemia in the fetus is a typical challenge for the obstetricians. The patients occasionally suffer from neurological injury following cerebral hypoxemia. The hypoxic insult may also affect the respiratory activity significantly. For example, acute alveolar hypoxia causes pulmonary vasoconstriction by damaging pulmonary vascular smooth muscle (i) and results in reduction of fatty acid oxidation by limiting the ATP supply required for metabolic processes (2). Hypoxia has also been shown to decrease the rate of palmitate incorporation into phospholipids (3), inhibit rate of fatty acid synthesis (3) and depress rate of incorporation of fatty acid and phosphatidic acid into lipids (4). Despite the fact that fatty acids represent a major substrate for energy metabolism in lung, no work has been done on the fatty acid metabolism in fetal lung. The present study was designed to determine the fate of fatty acid oxidation in fetal lung during ischemic challenge. The levels of acyl CoA and acylcarnitine intermediates were also measured in order to determine the rate-controlling steps of fatty acid metabolism in the fetal lung. Materials and Methods Animals and Ischemic Experiments Pregnant white New Zealand rabbits were obtained from Camm Research Co., New Jersey. Experiments were performed during the periods of 25 to 28 days' of gestation. Ischemia was produced for various intervals of time by the inflation of aortic balloon with total reduction of uterine blood flow. In some experiments, the cause of ischemia was removed after 20 min., and 2 hr stabilization allowed before removing the fetuses. To study the effect of ischemia on lipid metabolism, fetuses were injected with (U-14C)-pa]mitate and 3H20 before 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

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Effect of Ischemia on Lipid Metabolism

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inducing the ischemia. Only the surviving fetuses were used in our study. Fetuses were killed immediately by decapitation, and the lungs were quickly removed. Tissue Analysis The lungs of the fetuses were quickly frozen by freeze-clamping at liquid nitrogen temperature. A part of the frozen tissue was deproteinized and used for the assay of lactate and pyruvate. Lactate and pyruvate were quantified by fluorimetric assay methods according to Passonneau and Lowry (5) and Passonneau (6) respectively. ATP, ADP and AMP were also determined by fluorimetric assay according to standard enzymatic methods after extracting the tissue with perchloric acid in ethanol at -20°C (7). A part of the frozen tissue was powdered, extracted with ice-cold perchloric acid (6%, w/v) and then centrifuged at 0°C. The supernatant was neutralized with KOH and used for the estimation of tissue content of free CoA, acetyl CoA, free carnitine and acetyl carnitine. Levels of long chain acyl CoA and acylcarnitine were estimated after alkaline hydrolysis of the washed perchloric acid precipitate (7). Free CoA was determined by using the ~ - k e t o g l u t a r a t e dehydrogenase reaction (8) and the combination of this reaction with carnitine : acetyl CoA transferase reaction provided a method of assaying free carnitine levels (9). Acetyl CoA and acetyl carnitine were assayed fluorimetrically according to Herrera et al (i0) and Pearson et al (9), respectively. Levels of the long chain acyl CoA and acyl carnitine were assayed as free CoA and carnitine after hydrolysis as described by Williamson and Corkey (7). Long chain acyl CoA was hydrolyzed at pH ]1.9 for 15 min at 55°C in the presence of i0 mM dithiothreitol. Long chain acylcarnitine was hydrolyzed at pH 12.9 for 2 hr at 70°C. The hydrolyzates were centrifuged at 0°C and the supernatants were neutralized with KOH and assayed as before. NAD and NADH were estimated in the mitochondrial fraction by measuring the increase in fluorescence after addition of yeast alcohol dehydrogenase plus ethanol and by measuring the decrease in fluorescence after addition of beefheart lactate dehydrogenase plus pyruvate, respectively (ii). A part of the frozen lung tissue was powdered at liquid nitrogen temperature and extracted with a mixture of chloroform and methanol (2 : i, v/v). The solvent was evaporated under nitrogen, and then the extract was dissolved in aminimal volume of chloroform. Silicic acid column chromatography (Bio-Sil HA, 325 mesh) was performed to separate neutral lipids and phospholipids. The neutral lipids were washed with two volumes of chloroform, evaporated under nitrogen and saponified by heating at 60oc for 30 min after adding 4% KOH in 95% ethanol, and their contents were estimated by measuring glycerol release according to the method described by Garland and R a m i e (12). Estimation of Isotopic Palmitate and Water Incorporation Into Lipid The rate of in vivo lipogenesis in fetal lung was measured by determining the incorporation of tritium from 3Ho0 into fatty acids according to the method of Lowenstein et al (13). Incorporation of (U-14C)-palmitate into lipid was determined by counting a fraction of chloroform elute from silicic acid column as described above.

Results Effect of Ischemia on the Lactate and Pyruvate Content The lactate content of the fetal lung doubled after the ischemic insult,

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but decreased by a small amount after a 2 hr stabilization period (Table I). A small increase was noticed in the amount of pyruvate in the ischemic lung. However, the lactate/pyruvate ratio increased 1.7-fold during ischemia, and, came down to normal levels after the stabilization. TABLE I Effect of Ischemia on the Lactate and Pyruvate Content of Fetal Lung

Control

Ischemica

Ischemia Followed By 120 Min. Stabilization b

umol/gm dry wt. c Lactate

4.92 ± 0.35*

Pyruvate

0.14 ± 0.05**

Lactate Pyruvate

Ratio

35.1

± 1.62"

10.15 ± 1.51" 0.17 ± 0.44** 59.7

± 3.28*

5.26 ± 0.43 0.16 ± 0.06 32.9

± 2.0

a Ischemia was produced by the inflation of aortic balloon with total reduction of uterine blood flow for 20 min. b 120 minute stabilization was allowed after the ischemic period c Each value represents the Mean ± SE for 7 sets of experiments, each set containing 6 to 12 fetuses * p < 0.001

** p < 0.05

Effect of Ischemia on the Adenine Nucleotide Content Adenine nucleotide levels of the fetal lung are shown in Table II. Total adenine nucleotide content decreased to 72% of control after ischemia. The amount of ATP also decreased resulting in a large reduction of ATP/ADP ratio. Energy charge defined by (ATP + ½ADP)/ (ATP + ADP + AMP) also reduced to 86% of control during ischemia. The two hour stabilization resulted in restoring these values to the normal levels. Effect of Ischemia on the Levels of Acyl-CoA and Acyl-Carnitine Derivatives To determine the effects of ischemia on the rate-controlling steps of fatty acid metabolism, the levels of acyl CoA and acylcarnitine derivatives were measured'after inducing ischemia. The levels of CoA, acetyl CoA, carnitine and acetyl carnitine decreased significantly while those of long chainacyl CoA and long chain acyl carnitine enhanced (Table IIl). The tissue levels of acyl CoA and acylcarnitine derivatives returned to normal after 2 hour stabilization. Effect of Ischemia on the Levels of NADH and NAD The shown in ischemia lization

levels of NADH and NAD in the normal and ischemic fetal lungs are Table IV. The tissue levels of NADH increased 1.6-fold during resulting in a dramatic increase in NADH/NAD ratio. 2 hour stabireturned this ratio to normal value.

572

Effect

of Ischemia

on Lipid M e t a b o l i s m

TABLE Effect

of Ischemia

on the Adenine

Control

33, No. 6, 1983

II

Nucleotide

Ischemic a

umol/gm

Vol.

Content

of Fetal

Ischemia 120 Minute

Lung

Followed By Stabilization b

dry wt. c

Total Adenine Nucleotide

9.88 ± 0.76*

7.20 ± 0.85*

9.02 ± 0.91

ATP

8.46 ± 0.57**

4.86 ± 0.63**

7.95 ± 0.87

ADP

1.]2 ± 0.18

1.70 ± 0.12

1.05 ± 0.08

.AMP

0.30 ± 0.05

0.64 ± 0.16

0.32 + 0.09

ATP/ADP

7.55 ± 0.70**

2.86 ± 0.22**

7.57 + 0.66

ATP + ½ ADP ATP + ADP + AMP

0.913±

0.793±

0.909± 0.02

a Ischemia was produced tion of uterine blood b 120 minute

0.01"*

0.02**

by the inflation of aortic flow for 20 minutes.

stabilization

was allowed

after

the ischemic

c Eachv~ue represents the Mean ± SE for 7 sets containing 6 to 12 fetuses. * p < 0.05 Effect

of Ischemia

on M e t a b o l i s m

balloon

with

total

reduc-

period.

of experiments

each set

** p < 0.001 of Lipids

Ischemia caused a small increase in neutral Jipid content of fetal lungs (Fig i). The lipid content increased p r o g r e s s i v e l y with the duration of ischemia. I n c o r p o r a t i o n of (U-14C)-palmitate into lipid and 3H20 into fatty acids showed a linear relationship with time in the control lungs. (Figure 2 and 3). Ischemia caused enhanced incorporation of isotopic pa]mitate (Figure 2) and H20 (Figure 3) with time. Effect of Occlusion

of the Aorta

on the Dry Wt of the Lun~

After the occlusion of the aorta, lungs of the fetuses were dried to a constant weight. Occlusion of the aorta did not alter the dry wt/wet wt ratio of the lungs by any significant amount.

Discussion From the data presented in our study, it appears that B-oxidation is inhibited in the ischemic fetal lungs. In addition, increase in the levels of long chain acyl CoA and a c y l c a r n i t i n e with the concomitant decrease in CoA, a c e t y l C o A and a c e t y l c a r n i t i n e indicates that B-oxidation becomes rate-limiting in fatty acid metabolism. Under the normal conditions, B-oxidation does not appear to control the fatty acid oxidation in the m a m m a l i a n system (14, 15). The significant rise in NADH/NAD ratio during the ischemic challenge might

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Effect of Ischemia on Lipid Metabolism

573

TABLE III Effect of Ischemia on the Levels of Acyl CoA and Acylcarnitine Derivatives in Fetal Lung

Control

Ischemica

Ischemia Followed By 120 Min Stabilization b

umol/gm/dry wt c CoA

0.075 ± 0.005*

0.052 ± 0.003*

0.071± 0.004

Acetyl CoA

0.Ii

± 0.03**

0.03

± 0.005**

0.09 ± 0.01

Long Chain Acyl CoA

0.16

± 0.02*

0.30

± 0.03*

0.18 ± 0.02

Carnitine

1.52

± 0.ii*

0.90

± 0.08*

1.35 ± 0.12

Acetyl Carnitine

0.18

± 0.03**

0.05

± 0.002**

0.16 ± 0.02

Long Chain Acyl Carnitine

0.12

± 0.02*

0.38

± 0.07*

0.14 ± 0.03

a Ischemia was produced by the inflation of uterine blood flow for 20 minutes. b 120 minute stabilization

was allowed

of aortic balloon with total reduction

after the ischemic

period.

c Each value represents the Mean ± SE for 7 sets of experiments, taining 6 to 12 fetuses. * p< 0.01 * p< 0.005

each set con-

TABLE IV Effect of Ischemia on the Levels Control

of NADH and NAD in Fetal Lung Ischemic a

Ischemia Followed By 120 Min Stabilizationb

umol/gm dry lung c NADH

0.32 i 0.03*

0.50 ± 0.05*

0.36 i 0.02

NAD

1.5

i.i

1.62 ± 0.08

NADH/NAD

0.21 ± 0.04*

± 0.Ii**

± 0.07**

0.45 ± 0.03*

0.22 ± 0.03

a Ischemia was produced by the inflation of aortic balloon with total reduction of uterine blood flow for 20 minutes. b 120 minute stabilization was allowed after the ischemic period. c Each value represents the ~ean ± SE for seven sets of experiments containing 6 to 12 fetuses. * p< 0.005

** p< 0.01

each set

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Effect of Ischemia on Lipid Metabolism

Vol. 33, No. 6, 1983

10 O z D .._,1

-/

8

,~

~-}

ISCHEMIC

O~.

~}NOR~L Z'~

n

2

0

I

i

t

5 10 DURATION OF ISCHEMIA

o

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15

20

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Fig. i. The lipid content in normal and ischemic lung as a function of time. Each fetus in each litter was injected with i00 uCi of (U-14C) palmitate before the ischemic challenge. Control rabbits were not subjected to ischemic insult. Fetuses were sacrificed at indicated time intervals and lungs were quickly removed. Absolute levels of neutral lipids were measured as micromoles of glycerol released after extraction and hydrolysis. The number of sets in each group is indicated by the value in parentheses. have caused this inhibition of B-oxidation. The ischemic lungs of the rabbit fetuses showed enhancement of lactate/ pyruvate ratio and reduction of ATP/ADP ratio. Our results are in agreement with the previous reports where the same observation has been reported using hypoxic rat lungs (2). These investigators also noticed an increase in the elongation of the newly synthesized fatty acids in hypoxic lungs presumably as a result of the reduced rate of 6-oxidation. a E ..~

.t2 ~~ISC.HEMIC

O

5

10 DURATION OF ISCHEMIA minutes

15

20

Fig. 2. The incorporation of isotopic palmitate in normal and ischemic lung as a function of time. Each fetus in each litter was injected with i00 mCi of (U-14C)palmitate before the ischemic challenge. Control rabbits were not subjected to ischemic insult. Fetuses were sacrificed at indicated time i n t e r v ~ s and lungs were quickly removed. Incorporation of radiolabeled carbon from (U-14C)palmitate into lipid was calculated as micromole equivalents of palmitate incorporated per umole of lipid. The number of sets in each group is indicated by the value in parentheses.

Vol. 33, No. 6, 1983

Effect of Ischemia on Lipid Metabolism

575

.3O Q.

~0

~

.25

6

ISCHEMIC

z

0

.20

I

Z

2 ° §z . o s 00

10

DURATIONOFISCHEMIA minutes

1,5

20

Fig. 3. Pulmonary lipogenesis in normal and ischemic lung as a function of time. Each fetus in each litter was injected with 2 mCi of 3H20 before the ischemic challenge. Control rabbits were not subjected to ischemic insult. Fetuses were sacrificed at indicated time intervals and lungs were quickly removed. The radioactivity was determined from isolated lipid fractions. The number of sets in each group is indicated by the value in parentheses. Hypoxia accelerates the rate of lipogenesis in liver and heart of rat (16,17). We also observed increased amount of long chain acyl CoA (substrate for the lipid synthesis) and enhanced lipid content in the ischemic fetal lungs compared to the normal controls. These conditions might be expected to result in increased rates of the synthesis of lipids. Indeed, this was supported from the observations of the enhanced lipogenesis in ischemic lung. Incorporation of isotopic palmitate into the lipid was also increased. Similar to our study, increased lipogenesis in conjunction with the increased lactate content was previously reported for the mouse liver (18). In some of our experiments, we allowed 2 hour stabilization after 20 minutes of ischemic challenge before removing the fetus. The ratios of lactate/pyruvate, ATP/ADP, and NADH/NAD returned to the corresponding control levels after the cause of ischemia was removed. The levels of acyl CoA and acylcarnitine derivatives, however, remained unchanged even after 2 hr of stabilization. The long term accumulation of long chain acyl derivatives may have severe consequences on the fatty acid metabolism in fetal lungs. High levels of long chain acylcarnitine inhibits (Na +, K+)-ATPase activity (19). Reduced rate Of fatty acid activation might also cause the inhibition of (Na +, K+)-ATPase activity presumably by increasing the tissue levels of free fatty acids. Moreover, enhanced levels of acyl CoA and acylcarnitine may result generalized detergent effects which in turn may disrupt the lung membranes.

References i. 2. 3. 4. 5. 6.

A. P. FISHMAN, Circ. Res. 38, 221-231 (1976). D. J. P. BASSETT, A. B. FISHER and J. L. RABINOWITZ, Am. J. Physiol. 227, 1103-1108 (1974). A. NAIMARK and D. KLASS, Can. J. Physiol. Pharmacol. 45, 597-607 (1967). D. NEWMAN and A. NAIMARK, Am. J. Physiol. 214, 305-312 (1968). J. V. PASSONNEAU and O. H. LOWRY, In H. U. Bergmeyer ed, Methods of Enzymatic Analysis, New York, Academic Press, Vol 3, p 1452 (1970). J. V. Passonneau, In H. U. Bergmeyer ed, Methods of Enzymatic Analysis,

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7. 8. 9. I0. ii.

12. 13. 14. 15. 16. 17. 18. 19.

Effect of Ischemia on Lipid Metabolism

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New York, Academic Press, Vol 3, p 1468 (1970). J. R. WILLIAMSON and B. CORKEY, In J. Lowenstein eds, Methods of Enzymology, New York, Academic Press, Vol 13, p 434 (1969). P. B.GARLAND, D. SHEPHERD and D. W. YATES, Biochem. J. 97, 587-594 (1965). D. J. PEARSON, J. F. A. CHASE and P. K. TUBBS, In J. M. Lowenstein eds, Methods of Enzymology, New York, Academic Press, Vol 14, p 612 (1969). E. HERRERA and N. J. FREINKEL, J. Lipid Res. 8, 515-518 (1967). R. W. ESTABROOK, J. R. WILLIAMSON, R. FRENKEL and P. K. MAITRA, In R. W. Estabrook and M. E. Pullman ed, Methods of Enzymology, N.Y., Academic Press, Vol i0, p 474 (1967). P. B. GARLAND and P. J. RANDLE, Nature 196, 987-988 (1962). J. M. LOWENSTEIN, H. BRUNENGRABER and M. WADKE, In J. M. Lowenstein ed, Methods of Enzymology, New York, Academic Press, Vol 35, p 279 (1975). J. F. ORAM, J. I. WENGER and J. R. NEELY, J. Biol. Chem. 250, 73-78 (1975) D. W.YATES and P. B. GARLAND, Biochem. J. 119, 547-552 (1970). J. R. EVANS, Circ. Res. 15, 96-106 (1964b). V. L. KINNULA, M. O. SAVOLAINEN and I. HASSINEN, Acta. Physiol. Scan. 104, 148-155 (1978). D. M. W. SALMON, N. L. BOWEN and D. A. HEMS, Biochem. J. 142, 611-618 (1974). J. M. WOOD, B. BUSH, B. J. R. PITTS and A° SCHWARTZ, Biochem. Biophys. Res. Commun. 74, 677-684 (1977).