108
Biochimica et Biophysica Acta, 444 (1976) 108--117 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27962
E X T R A C E L L U L A R ASCORBIC ACID IN LUNG
R.J. WILLIS and C.C. KRATZING
Physiology Department, University of Queensland, St. Lucia 406 7 (Australia) (Received December 18th, 1975)
Summary Fifty percent of the ascorbic acid content of sliced rat lung was released from the tissue to the media within a few minutes by either washing or incubating the slices with Krebs-phosphate solution. Measurement of the lactate dehydrogenase and potassium content of the medium after incubating lung slices for 5 min showed that about 20% of the cells were damaged by slicing. Sephadex chromatography of tissue extracts prepared from washed lung slices showed that none of the ascorbic acid in these slices was bound to protein. Also, metabolic poisons were shown to deplete the ascorbic acid content of washed lung slices. Approx. 57% of the lung ascorbic acid of guinea pigs that had been supple° m e n t e d with ascorbic acid and 78% of the lung ascorbic acid of ascorbic aciddeficient guinea pigs were found in the medium when lung slices from these animals were incubated with Krebs-phosphate solution. These results were taken to indicate the presence of an extracellular pool of ascorbic acid in lung which is maintained even during scurvy.
Introduction Ascorbic acid is transported into the lung in the reduced form [1], where it appears to accumuYate in the fluid lining the air spaces [2]. The function of ascorbic acid at this site may be to act as an extracellular antioxidant, protecting some quality of either surfactant or mucus. The present paper reports on the characterization of the different ascorbic acid compartments in rat lung and examines the size of the extracellular ascorbic acid c o m p a r t m e n t in guinea pig lung during the course of scurvy.
109 Materials and Methods
Animals Male rats (150--200 g) and guinea pigs (200--400 g) were of the Queensland University Central Animal House strain. Rats were fed a commercial breeding ration and unless otherwise specified guinea pigs were maintained on a diet of dried lucerne, breeding pellets and a supplement of ascorbic acid. Rat experiments Rats were anaesthetized with pentobarbitone (280 pmol/kg), killed and exsanguinated by cutting the vena cava. Lungs were excised, and after removal of the extrapulmonary vessels, were cut into 0.5 mm thick slices. These were obtained by placing tissue in the elbow of a multi-wire frame which was then slowly drawn through a razor-blade grid with a slicing action. Slices were washed in a nylon net by dripping Krebs-phosphate solution at 37°C onto the tissue at rates ranging from 1.3 to 7.9 ml/min. For this purpose Krebs-phosphate solution was pumped at constant flow through a heat exchanger at 37°C and onto the tissue. The whole apparatus was enclosed in a plexiglass box to help maintain the temperature and a high humidity. In the nylon net, tissue slices were suspended in a small pool of fluid that was mixed each time a drop of Krebsphosphate solution fell. The effluent from the net was collected in serial 3 ml fractions. For other experiments, slices were washed in this apparatus for 10 min with a washing rate of 4.6 ml/min. To estimate the degree of cellular damage caused by slicing, lung slices were incubated with 3 ml of 0.2% (w/v) glutathione with Krebs-phosphate solution in a metabolic shaker at 37°C. Glutathione was used to prevent the loss of ascorbic acid by oxidation. After 5 min, tissue and medium were separated using a nylon net and assayed for ascorbic acid [3], DNA [4], protein [5] and lactate dehydrogenase (E.C. 1.1.1.27} activity [6]. Potassium was estimated by flame p h o t o m e t r y . The oxygen consumption of slices incubated at 37°C with 0.2% (w/v) glutathione in Krebs-phosphate solution was measured over 1 h using a Warburg manometer [7] and the total lactate content of tissue and medium was estimated [8] before and after this incubation period. The uptake of ascorbic acid by washed lung slices was examined by incubating them in 0.2% {w/v) glutathione in Krebs-phosphate solution that contained 0, 20, 40, 80, 160, or 320 pg of ascorbic acid/ml. After 2, 4, 10, or 30 min, tissue and medium were separated and assayed for ascorbic acid and DNA. To test if the ascorbic acid that remained in the tissue slices after washing was protein bound, a 25% (w/v) homogenate of washed lung slices in 154 mM potassium phosphate buffer (pH 6.5) that contained 1.2 mM MgSO4 and 0.2% (w/v) glutathione was prepared in a glass-teflon homogenizer. After centrifuging this homogenate at 8500 X g for 15 min, 1 ml of the supernatant was added to a Sephadex G-25 column (1 cm X 40 cm; void volume, 14.5 ml) that was equilibrated and developed with the phosphate buffer at a flow rate of 0.45 ml/min at 4°C. Serial fractions were collected and assayed for protein and ascorbic acid. To examine the effect of metabolic poisons on the residual ascorbic acid c o n t e n t of slices after washing, washed lung slices were incubated at 37°C with
110 0.2% (w/v) glutathione in Krebs-phosphate solution. In some experiments, the medium contained 2 mM cyanide and 10 mM fluoride, either singly or together. After incubation for 1 h, slices were separated from the medium by filtering through a nylon net and both slices and media were assayed for ascorbic acid and DNA.
Guinea pig experiments Guinea pigs were given either no oral ascorbic acid (supp. 0) or supplements of 2 mg (supp. 2) or 10 mg (supp. 10) of ascorbic acid/100 g/day. After 20 days, they were anaesthetized, killed and exsanguinated, and their lungs were excised, sliced and assayed for ascorbic acid, protein and DNA. Other lung slices were incubated with 3 ml of Krebs-phosphate solution that contained 0.2% (w/v) glutathione in a metabolic shaker at 37 ° C. After 1 h, slices and medium were separated with a nylon net then assayed. Extraction and chemical assay Fresh lung slices, or slices which have been washed or incubated were blotted firmly to remove adhering fluid and suspended in 5% (w/v) metaphosphoric acid for 1 h to extract ascorbic acid and precipitate protein. After centrifuging at 1000 X g for 10 min, extracts were estimated for ascorbic acid and the denatured tissue pellets were assayed for DNA and protein. Incubation media and washings were assayed in a similar manner after the addition of 25% (w/v) metaphosphoric acid. For the preparation of tissue extracts for the lactate dehydrogenase assay, tissue slices were homogenized with 50-times their weight of 0.05 M sodium phosphate buffer (pH 7.4) using a glass-teflon homogenizer, and the homogenate was centrifuged at 1000 × g for 15 min. Media were prepared in a similar fashion with the exception that no buffer was added prior to homogenization. Potassium was extracted from tissue with 1 N nitric acid after ashing in a platinum crucible at 600°C for 12 h. The medium potassium was estimated directly w i t h o u t preparation. The inulin space was estimated by incubating washed lung slices with 0.1% (w/v) inulin in Krebs-phosphate solution at 37°C. After 1 h, slices were separated from the medium, blotted, and extracted twice with 5% (w/v) metaphosphoric acid, and 1 ml of 25% (w/v) metaphosphoric acid was added to the media. Both tissue and media extracts were assayed for inulin [9]. Results
Rat experiments The rate of removal of ascorbic acid from lung slices increased with the washing rate until at¢7.9 ml/min a maximum rate of ascorbic acid removal was obtained (Fig. 1). At this washing rate, all the ascorbic acid that could be removed from the tissue was washed out within 2 min. Similar rates of removal were found for both DNA and protein. Of the 22.7 pg of ascorbic acid/mg of DNA originally present in the tissue, about half remained in the tissue slices and the rest was recovered in the wash {Table I). Washing also removed 7.3%
111
100
75
2.2
50
4.6
25
O
5
10 15 Time {min)
20
F i g . 1. T h e e f f e c t o f w a s h i n g r a t e o n t h e r e m o v a l o f a s c o r b i c a c i d f r o m l u n g s l i c e s . V a l u e s a r e t h e p e r c e n t of the total ascorbic acid washed out of the slices.
of the total DNA in the tissue and 27.1% of the total protein. During prolonged washing, the tissue ascorbic acid c o n t e n t fell from 23.0 ± 1.3 (3) pg/mg DNA to 12.1 ± 0.8 (3) pg/mg DNA in 10 min. After a further 170 min only a small additional decrease to 10.6 + 0.5 (3) pg of ascorbic acid/mg of DNA was measured. When lung slices were incubated for 5 min in a shaker, 51.5% of the original tissue content of ascorbic acid and 29.3% of the protein were recovered in the medium (Table II). The proportion of lactate dehydrogenase activity and potassium released was 17.1% and 22% respectively. Sliced lung that had first been perfused with saline via the pulmonary artery, released 17.2% of its protein. About 6% of the original DNA content of lung slices was recovered in the medium irrespective of whether or not the lungs were perfused prior to slicing. Lung slices had an oxygen consumption of 8.68 ± 0.16 (4) pl/mg dry wt./h and an aerobic lactate production of 0.45 -+ 0.04 (4) pg/mg dry wt./h. Incubation of washed slices with media containing 0--320 pg of ascorbic acid/ml, produced m a x i m u m tissue levels of ascorbic acid within 2 min, after which little change occurred in the next 28 min. Equilibrium values in this experiment show a linear relation between the medium ascorbic acid concentration and the tissue ascorbic acid content (Fig. 2). Centrifugation of homogenized washed lung slices produced supernatants that contained 84.0 ± 3.7 (5) % of the total (supernatant + pellet) ascorbic acid, 4.4 t 1.6 (4) % of the total DNA, and 52.3 + 5.7 (4) % of the protein. After chromatography on G-25 Sephadex, a clear separation of the protein and TABLE
I
THE PROPORTION OF THE ORIGINAL ASCORBIC LUNG SLICES THAT IS REMOVED BY WASHING V a l u e s a r e m e a n s +-S.E. w i t h t h e n u m b e r
Fresh lung Washed lung R e c o v e r e d in w a s h
of observations
ACID,
DNA,
AND
PROTEIN
shown in parenthesis.
Ascorbic acid (~zg/mg D N A )
DNA (% of original)
Protein (% o f o r i g i n a l )
2 2 . 7 + 1.1 ( 5 ) 10.0 ± 0.6 (5) 12.1 + 0.5 (4)
-9 2 . 7 + 1.9 ( 4 ) 7.3 + 1.9 (4)
-72.9 ± 3.7 (4) 27.1 ± 3.7 (4)
CONTENT
OF
112
T A B L E II THE RELEASE OF TISSUE CONSTITUENTS UM AFTER 5 MIN
FROM
LUNG SLICES INTO THE INCUBATION
MEDI-
V a l u e s are m e a n s o f f o u r o b s e r v a t i o n s -+S.E. T h e r e s u l t s axe f o r s l i c e s o f f r e s h l y e x c i s e d l u n g . ' P e r f u s e d lung' means that the intact lung was perfused via the pulmonary artery before slicing. Tissue constituent
Percent of the initial tissue content f o u n d in the m e d i u m
Ascorbic acid Protein Lactate dehydrogenase activity Potassium Protein (perfused lung) DNA DNA (perfused lung)
51.5 29.3 17.1 22.0 17.2 6.8 6.3
+ 2.2 + 2.3 -+ 0 . 6 -+ 2 . 0 + 1.9 + 0.8 + 0.6
both the reduced and oxidized ascorbic acid was obtained (Fig. 3). Incubation of washed lung slices with fluoride, cyanide, or fluoride and cyanide together, produced decreases in the tissue ascorbic acid content of 19%, 28%, and 44% respectively (Table III). In this experiment, ascorbic acid that was lost from the tissue could be recovered in the medium. The inulin space was estimated to be 0.20 + 0.03 (6) ml/g wet weight of tissue and the plasma ascorbic acid concentration was measured to be 14.0 _+ 1.6 (9) pg/ml.
Guinea pig experiments Guinea pigs fed the ascorbic acid-deficient diet (supp. 0) lost weight after about the twelfth day and died approximately 10 days later, showing acute 30
~-
20
20'/0 I n u l i n
?g
space
o
161 i-=
I
I
I
100 200 300 Medium ascorbic acid ~' (pg/ml)
i
400
F i g . 2. A s c o r b i c a c i d c o n t e n t o f t i s s u e a n d m e d i u m a t e q u i l i b r i u m . V a l u e s are m e a n s w i t h t h e n u m b e r o f o b s e r v a t i o n s s h o w n i n p a r e n t h e s i s . T h e d o t size c o v e r s -+S.E. T h e r a p i d i t y w i t h w h i c h a s c o r b i c a c i d c a n a c c u m u l a t e i n t i s s u e w h e n s l i c e s are i n c u b a t e d w i t h m e d i a c o n t a i n i n g a s c o r b i c a c i d s u g g e s t s t h a t i t is t h e i n t e r s t i t i a l f l u i d s p a c e t h a t is f i l l e d w i t h a s e o r b i c a c i d . H o w e v e r , s o m e d i f f u s i o n i n t o t h e t i s s u e c o m p a r t ment must have occurred since calculation based on an inulin space of 0.2 ml/g of tissue shows that only p a r t o f t h e tissue levels of a s c o r b i c acid at e q u i l i b r i u m can be a c c o u n t e d for on this basis.
113
15
15
.-& 10 E
~ ~ L o_
10 ~ c) o (3-
Protein
AH2
5 S.
5
i
5
0
15
Fraction
20 25 number
Fig. 3. S e p h a d e x ( G - 2 5 ) s e p a r a t i o n Oxidized ascorbic acid, (A).
30
35
o f an e x t r a c t o f w a s h e d l u n g slices. R e d u c e d a s c o r b i c a c i d , ( A H 2 ) ;
signs of scurvy. At 20 days, the organ ascorbic acid levels were found to be negligable in comparison to those of supp. 10 guinea pigs. In the incubation experiments, 57, 58 and 78% of the original ascorbic acid content of lung slices from supp. 10, supp. 2, and supp. 0 guinea pigs respectively were found in the medium following incubation with Krebs-phosphate solution (Table IV). Slices from supp. 0 guinea pigs released about twice the amount of D N A into the medium than did slices from supp. 10 animals and they released about 9% more protein. Supp. 2 slices lost intermediate quantities of D N A and protein. Discussion Extracellular ascorbic acid in rat lung The rapid removal of about half the ascorbic acid content of lung slices by washing suggests that this ascorbic acid may be extracellular. The loss of whole cells and the contents of damaged cells will contribute some ascorbic acid to
T A B L E III THE EFFECT OF FLUORIDE LUNG SLICES
AND CYANIDE ON THE ASCORBIC ACID CONTENT OF WASHED
V a l u e s are m e a n s o f t h r e e o b s e r v a t i o n s ± S . E . M e a n s w i t h like s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y u s i n g S t u dent's t test with P < 0.05. Treatment
T i s s u e a s c o r b i c acid c o n t e n t (pg/mg DNA)
F r e s h l u n g slices W a s h e d l u n g slices a f t e r i n c u b a t i o n Fluoride (10 raM) C y a n i d e (2 r a M ) Fluoride and cyanide
22.98 10.27 8.35 7.43 5.78
+ ~ * ± ±
0.33 0.17 0.59 0.22 0.20
a abcd b c d
114 T A B L E IV THE DISTRIBUTION OF ASCORBIC ACID, DNA, AND PROTEIN BETWEEN THE TISSUE AND MEDIUM AFTER INCUBATION OF LUNG SLICES FROM ASCORBIC ACID-SUPPLEMENTED AND ASCORBIC ACID-DEFICIENT GUINEA PIGS IN KREBS-PHOSPHATE FOR 1 h V a l u e s are m e a n s +-S.E. w i t h t h e n u m b e r o f o b s e r v a t i o n s s h o w n i n p a r e n t h e s i s . Ascorbic acid supplement (rag/100 g/day)
Tissue ascorbic acid (/zg/mg DNA)
Medium ascorbic acid (pg/mg DNA)
Medium DNA (% o f t o t a l )
Medium protein (% o f t o t a l )
10 2 None
6 . 9 6 ÷ 0 . 5 7 (S) 0.80 + 0.06 (16) 0 . 2 1 ± 0 . 0 4 (5)
9 . 2 8 ÷ 0 . 9 0 (8) 1.09 ~ 0.09 (16) 0 . 6 3 ~ 0 . 0 3 (5)
9.0 t 0.9 (8) 12.1 + 1.3 ( 1 6 ) 2 0 . 1 4 1.6 ( 7 )
3 5 . 7 " 1.9 (8) 4 0 . 3 ~ 1.3 ( 1 6 ) 4 4 . 6 + 0 . 6 (7)
the total a m o u n t washed out of the slices, but the size of this cont ri but i on is difficult to estimate. When slices are incubated for 5 min, about half of the original tissue ascorbic acid c o n t e n t can be accounted for in the medium and this agrees with the p r o p o r t i o n of ascorbic acid that is removed by washing. 7% o f the original tissue DNA is removed by either washing or incubating the slices and this probably represents the removal of free cells such as alveolar macrophages. Since lung slices were f o u n d to consume oxygen at a rate similar to those described previously for lung [10], and aerobic glycolysis was found to be a quarter of comparable values r e por t ed elsewhere [11], it was considered t h at only a small a m o u n t of cellular damage was caused by the present m e t h o d of slicing. Some of the relatively large a m o u n t of tissue protein that is removed by washing or incubation will be associated with the removal of cells and damaged cell contents, but a p r o p o r t i o n must have also been extracellular and present in the interstitial fluid space, the circulation, or the airspaces. The co n tri but i on of protein from the blood can be seen by the fact t h a t when tissue slices from the perfused lung were incubated, only 17% of the protein was f o u nd in the medium. A similar percentage of the activity of the cytoplasmic en zym e , lactate dehydrogenase, also appeared in the medium. Ano t h er estimate o f cell damage is that 22% of the tissue potassium is found in the medium when slices are incubated. Part of this potassium would be associated with e r y t h r o c y t e s and some would have been extracellular. The potassium co n cen tr atio n in the respiratory tract fluid of man is 13 mM [12] and it m a y be higher in rat since in the submaxillary-sublingual gland secretions of this species, its concentration is about 50 mM [13]. From these results, it is concluded that the e x t e n t of cell loss and damage in the lung slices is about 20%. Th e rapid depletion of an intracellular c o m p a r t m e n t of ascorbic acid by diffusion might also account for the removal of some tissue ascorbic acid by washing, but this is unlikely because the original tissue c o n t e n t of ascorbic acid could n o t be reconstituted by incubating washed tissue slices in media that contained ascorbic acid in excess of the plasma concentration. Th e ready removal of about half the ascorbic acid, c o n t e n t of lung slices by either washing or incubation procedures, taken in conjunction with an estimate o f 20% for the tissue damage caused by slicing, suggests that about 30% of the ascorbic acid in lung is extracellular. Almost none of this ascorbic acid is present in the interstitial fluid space because the plasma concent rat i on of ascorbic
115 acid is 14 pg/ml and the inulin space is 0.2 ml/g of tissue. The present findings agree with the result obtained previously that 30% of the ascorbic acid content of an intact lung can be removed by washing the airspaces [2], and supports the conclusion that ascorbic acid is concentrated in the fluid lining the respiratory epithelium. hztracellular ascorbic acid in rat lung After removal of the extracellular ascorbic acid by washing, the remainder is unaffected by further washing. This residual ascorbic acid may be contained in an actively concentrating compartment since incubation of washed slices with metabolic poisons decreased the tissue content of ascorbic acid and under the mild conditions of Sephadex chromatography, the ascorbic acid in washed lung slices was not found to be protein bound. In adrenal and liver tissue there is evidence that ascorbic acid is b o u n d to protein [14--16], although this has been questioned by Lewis et al. [17]. March and Tolbert [18] have found only non-specific binding of ascorbic acid in guinea pig brain homogenates. In the present experiments, no evidence of protein-bound ascorbic acid has been found in rat lung homogenates under the conditions of sephadex chromatography. Fig. 4 summarizes the characteristics of the ascorbic acid compartments in rat lung and shows how these compartments may interact. Extracellular ascorbic acid in guinea pig lung Lung slices from guinea pigs fed ascorbic acid (supp. 10 and supp. 2) released a b o u t 57% of their ascorbic acid content on incubation. Assuming the same degree of damage as in the rat experiments, about 37% of this ascorbic acid may be extracellular. In ascorbic acid-deficient guinea pigs, where the lung ascorbic acid content drops to about 1% of the original level, 78% of the ascorbic acid is readily removed by incubation. Of this 58% may be extracellular.
Plasma (pglm[)
;sue a/g}
Airspace
Airspace
fluid
(pg/mt)
14
g2
3602100
Fig. 4. P u l m o n a r y ascorbic acid c o m p a r m e n t s . Ascorbic acid f r o m the plasma is transported into a lung tissue c o m p a r t m e n t w h e r e it is concentrated. It is then m o v e d across the respiratory epithelium where it accumulates in the fluid lining the airspaces. Calculation of the values in the figure is based o n a lung ascorbic acid content of 2 4 0 pg/g J21 J of which 7 0 % is tissue ascorbic and 3 0 % is extracellular. It is assumed t h a t t h e airspace fluid h a s a v o l u m e o f f r o m 0 . 0 3 m l / g J2] t o 0 . 2 m l / g .
116
Although some of the extracellular ascorbic acid in the incubation media will be derived from oxidized ascorbic acid that is reduced by glutathione, it is suggested that the function of extracellular ascorbic acid in guinea pig lung is of sufficient importance for this ascorbic acid c o m p a r t m e n t to be maintained in p r o p o r t i o n to the intracellular pool of ascorbic acid, even in acute scurvy. Guinea pig (supp. 10) lung slices, like rat lung slices, release about 30% of their total protein and a bout 8% of their total DNA when t hey are incubated. However, when lung slices from suppl. 2 and suppl 0 guinea pigs are incubated there is an increase in the a m o u n t of protein and DNA released into the medium which is directly proportional to the deg-fee of ascorbic acid deficiency. A likely explanation is that since ascorbic acid is i m p o r t a n t in maintaining the intercellular matrix, it might be expect ed that cells will shake loose from ascorbic acid-deficient tissue slices on incubation. The increase in the a m o u n t of protein in the medium would thus be associated with the extra DNA released. A s c o r b i c acid as an extracellular a n t i o x i d a n t in lung
Since the lung is exposed to most of the atmospheric oxygen tension, it is attractive to propose that ascorbic acid might act as an antioxidant in this organ. In experiments where rats were exposed to hyperbaric oxygen [19] or ozone [20] administration of ascorbic acid prior to t r e a t m e n t lessened the toxic effects. Also, exposure to hyperbaric oxygen decreased rat lung ascorbic acid by 40% [21]. Since a b o u t 30% of the ascorbic acid c o n t e n t of rat lung appears to be extracellular and concent r a t e d in the fluid lining the airspaces, it may be proposed that it acts as an antioxidant at this site. Certainly there would seem to be a use for an extraceUular antioxidant in lung. Klaus et al. [22] showed th at the surface activity of an extract of surfactant from beef lung was gradually lost over 2 h if exposed to air. T hey concluded that for surfactant to resist oxygen at the air-tissue interface, it required the antioxidant potential o f substances as ye t unidentified. Also, exposure of snail mucus to o z o n e decreased the inhibition titer of the mucus with the hemagglutination test on the influenza viruses PR-8 and LEE [23]. It is concluded that a b o u t 30% o f the ascorbic acid c o n t e n t of rat and guinea pig lung may be acting as an extracellular antioxidant, protecting some p r o p e r t y of either surfactant or mucus. References 1 2 3 4 5 6 7 8 9 10 11 12 13
Willis, R . J . and Kratzing, C.C. ( 1 9 7 5 ) P f l u g e r s A r c h . 3 5 6 , 9 3 - - 9 8 Willis, R . J . and Kratzing, C . C . ( 1 9 7 4 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 5 9 , 1 2 5 0 - - 1 2 5 3 B o l i n , D.W. a n d B o o k , L. ( 1 9 4 7 ) S c i e n c e 1 0 6 , 4 5 1 Burton, K. (1956) Biochem. J. 62,315--323 G o a , J. ( 1 9 5 3 ) S c a n d . J . Clin. L a b . I n v e s t . 5 , 2 1 8 - - 2 2 2 W r 6 b l e w s k i , I. and Mason, H . S . ( 1 9 5 9 ) P r o c . S o c . E x p . Biol. M e d . 9 0 , 2 1 0 - - 2 1 3 U m b r e i t , W.W., B u r r i s , R . H . a n d S t a u f f e r , J . F . ( 1 9 6 4 ) M a n o m e t r i c T e c h n i q u e s , B u r g e s s P u b l i s h i n g C o . , Minnesota B a r k e r , S.B. a n d S u m m e r s o n , W . H . ( 1 9 4 1 ) J. Biol. C h e m . 1 3 8 , 5 3 5 - - 5 5 4 K u l k a , R . G . ( 1 9 5 6 ) B i o c h e m . J. 6 3 , 5 4 2 - - 5 4 8 B a r t o n , E . S . G . , Miller, Z.B. and Bartlett, G . R . ( 1 9 4 7 ) J . Biol. C h e m . 1 7 1 , 7 9 1 - - 8 0 0 L e v y , S.E. a n d H a r v e y , E. ( 1 9 7 4 ) J . A p p l . P h y s i o l . 3 7 , 2 3 9 - - 2 4 0 P o t t e r , J . L . , M a t h e w s , L . W . , L e m n , J . a n d S p e c t o r , S. ( 1 9 6 3 ) A n n . N . Y . A c a d . Sci. 1 0 6 , 6 9 2 - - - 6 9 7 S c h n e y e r , C . A . a n d S c h n e y e r , L . H . ( 1 9 5 9 ) P r o c . Soc. E x p . Biol. M e d . 1 0 1 , 5 6 8 - - 5 6 9
117 14 15 16 17 18 19 20 21 22 23
SumerwlU, W.N. and Sealock, R.R. (1952) J. Biol. Chem. 1 9 6 , 7 5 3 - - 7 5 9 Roe, J.H. and Itscoitz, S.B. (1963) Proc. Soc. Exp. Biol. Med. 1 1 3 , 6 4 6 - - 6 5 0 Fiddick, R. and Heath, H. (1967) Biochim. Biophys. Acta 1 3 6 , 2 0 6 - - 2 1 3 Lewis, W.H., Chiang, J.L. and Gross, S. (1960) Archs. Biochem. Biophys. 89, 21--26 March, S.C. and Tolbert, B.M. (1971) Fed. Proc. 30, 521 Jamieson, D. and van den Brenk, H.A.S. (1964) Biochem. Pharmac. 1 3 , 1 5 9 - - 1 6 4 Matzen, R.N. (1957) J. Appl. Physiol. 1 1 , 1 0 5 - - 1 0 9 Willis, R.J. and Kratzing, C.C. (1972) Am. J. Physiol. 222, 1391--1394 Klaus, M.H., Clements, J.A. and Havel, R.J. (1961) Proc. Natl. Acad. Sci. U.S. 47, 1858--1859 Falk, H.L., Kotin, P. and R o w l e t t e , W. (1963) Ann. N.Y. Aead. Sci. 106, 583--608
Biochimica et Biophysica Acta, 444 (1976) 108--117 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27962
E X T R A C E L L U L A R ASCORBIC ACID IN LUNG
R.J. WILLIS and C.C. KRATZING
Physiology Department, University of Queensland, St. Lucia 406 7 (Australia) (Received December 18th, 1975)
Summary Fifty percent of the ascorbic acid content of sliced rat lung was released from the tissue to the media within a few minutes by either washing or incubating the slices with Krebs-phosphate solution. Measurement of the lactate dehydrogenase and potassium content of the medium after incubating lung slices for 5 min showed that about 20% of the cells were damaged by slicing. Sephadex chromatography of tissue extracts prepared from washed lung slices showed that none of the ascorbic acid in these slices was bound to protein. Also, metabolic poisons were shown to deplete the ascorbic acid content of washed lung slices. Approx. 57% of the lung ascorbic acid of guinea pigs that had been supple° m e n t e d with ascorbic acid and 78% of the lung ascorbic acid of ascorbic aciddeficient guinea pigs were found in the medium when lung slices from these animals were incubated with Krebs-phosphate solution. These results were taken to indicate the presence of an extracellular pool of ascorbic acid in lung which is maintained even during scurvy.
Introduction Ascorbic acid is transported into the lung in the reduced form [1], where it appears to accumuYate in the fluid lining the air spaces [2]. The function of ascorbic acid at this site may be to act as an extracellular antioxidant, protecting some quality of either surfactant or mucus. The present paper reports on the characterization of the different ascorbic acid compartments in rat lung and examines the size of the extracellular ascorbic acid c o m p a r t m e n t in guinea pig lung during the course of scurvy.
109 Materials and Methods
Animals Male rats (150--200 g) and guinea pigs (200--400 g) were of the Queensland University Central Animal House strain. Rats were fed a commercial breeding ration and unless otherwise specified guinea pigs were maintained on a diet of dried lucerne, breeding pellets and a supplement of ascorbic acid. Rat experiments Rats were anaesthetized with pentobarbitone (280 pmol/kg), killed and exsanguinated by cutting the vena cava. Lungs were excised, and after removal of the extrapulmonary vessels, were cut into 0.5 mm thick slices. These were obtained by placing tissue in the elbow of a multi-wire frame which was then slowly drawn through a razor-blade grid with a slicing action. Slices were washed in a nylon net by dripping Krebs-phosphate solution at 37°C onto the tissue at rates ranging from 1.3 to 7.9 ml/min. For this purpose Krebs-phosphate solution was pumped at constant flow through a heat exchanger at 37°C and onto the tissue. The whole apparatus was enclosed in a plexiglass box to help maintain the temperature and a high humidity. In the nylon net, tissue slices were suspended in a small pool of fluid that was mixed each time a drop of Krebsphosphate solution fell. The effluent from the net was collected in serial 3 ml fractions. For other experiments, slices were washed in this apparatus for 10 min with a washing rate of 4.6 ml/min. To estimate the degree of cellular damage caused by slicing, lung slices were incubated with 3 ml of 0.2% (w/v) glutathione with Krebs-phosphate solution in a metabolic shaker at 37°C. Glutathione was used to prevent the loss of ascorbic acid by oxidation. After 5 min, tissue and medium were separated using a nylon net and assayed for ascorbic acid [3], DNA [4], protein [5] and lactate dehydrogenase (E.C. 1.1.1.27} activity [6]. Potassium was estimated by flame p h o t o m e t r y . The oxygen consumption of slices incubated at 37°C with 0.2% (w/v) glutathione in Krebs-phosphate solution was measured over 1 h using a Warburg manometer [7] and the total lactate content of tissue and medium was estimated [8] before and after this incubation period. The uptake of ascorbic acid by washed lung slices was examined by incubating them in 0.2% {w/v) glutathione in Krebs-phosphate solution that contained 0, 20, 40, 80, 160, or 320 pg of ascorbic acid/ml. After 2, 4, 10, or 30 min, tissue and medium were separated and assayed for ascorbic acid and DNA. To test if the ascorbic acid that remained in the tissue slices after washing was protein bound, a 25% (w/v) homogenate of washed lung slices in 154 mM potassium phosphate buffer (pH 6.5) that contained 1.2 mM MgSO4 and 0.2% (w/v) glutathione was prepared in a glass-teflon homogenizer. After centrifuging this homogenate at 8500 X g for 15 min, 1 ml of the supernatant was added to a Sephadex G-25 column (1 cm X 40 cm; void volume, 14.5 ml) that was equilibrated and developed with the phosphate buffer at a flow rate of 0.45 ml/min at 4°C. Serial fractions were collected and assayed for protein and ascorbic acid. To examine the effect of metabolic poisons on the residual ascorbic acid c o n t e n t of slices after washing, washed lung slices were incubated at 37°C with
110 0.2% (w/v) glutathione in Krebs-phosphate solution. In some experiments, the medium contained 2 mM cyanide and 10 mM fluoride, either singly or together. After incubation for 1 h, slices were separated from the medium by filtering through a nylon net and both slices and media were assayed for ascorbic acid and DNA.
Guinea pig experiments Guinea pigs were given either no oral ascorbic acid (supp. 0) or supplements of 2 mg (supp. 2) or 10 mg (supp. 10) of ascorbic acid/100 g/day. After 20 days, they were anaesthetized, killed and exsanguinated, and their lungs were excised, sliced and assayed for ascorbic acid, protein and DNA. Other lung slices were incubated with 3 ml of Krebs-phosphate solution that contained 0.2% (w/v) glutathione in a metabolic shaker at 37 ° C. After 1 h, slices and medium were separated with a nylon net then assayed. Extraction and chemical assay Fresh lung slices, or slices which have been washed or incubated were blotted firmly to remove adhering fluid and suspended in 5% (w/v) metaphosphoric acid for 1 h to extract ascorbic acid and precipitate protein. After centrifuging at 1000 X g for 10 min, extracts were estimated for ascorbic acid and the denatured tissue pellets were assayed for DNA and protein. Incubation media and washings were assayed in a similar manner after the addition of 25% (w/v) metaphosphoric acid. For the preparation of tissue extracts for the lactate dehydrogenase assay, tissue slices were homogenized with 50-times their weight of 0.05 M sodium phosphate buffer (pH 7.4) using a glass-teflon homogenizer, and the homogenate was centrifuged at 1000 × g for 15 min. Media were prepared in a similar fashion with the exception that no buffer was added prior to homogenization. Potassium was extracted from tissue with 1 N nitric acid after ashing in a platinum crucible at 600°C for 12 h. The medium potassium was estimated directly w i t h o u t preparation. The inulin space was estimated by incubating washed lung slices with 0.1% (w/v) inulin in Krebs-phosphate solution at 37°C. After 1 h, slices were separated from the medium, blotted, and extracted twice with 5% (w/v) metaphosphoric acid, and 1 ml of 25% (w/v) metaphosphoric acid was added to the media. Both tissue and media extracts were assayed for inulin [9]. Results
Rat experiments The rate of removal of ascorbic acid from lung slices increased with the washing rate until at¢7.9 ml/min a maximum rate of ascorbic acid removal was obtained (Fig. 1). At this washing rate, all the ascorbic acid that could be removed from the tissue was washed out within 2 min. Similar rates of removal were found for both DNA and protein. Of the 22.7 pg of ascorbic acid/mg of DNA originally present in the tissue, about half remained in the tissue slices and the rest was recovered in the wash {Table I). Washing also removed 7.3%
111
100
75
2.2
50
4.6
25
O
5
10 15 Time {min)
20
F i g . 1. T h e e f f e c t o f w a s h i n g r a t e o n t h e r e m o v a l o f a s c o r b i c a c i d f r o m l u n g s l i c e s . V a l u e s a r e t h e p e r c e n t of the total ascorbic acid washed out of the slices.
of the total DNA in the tissue and 27.1% of the total protein. During prolonged washing, the tissue ascorbic acid c o n t e n t fell from 23.0 ± 1.3 (3) pg/mg DNA to 12.1 ± 0.8 (3) pg/mg DNA in 10 min. After a further 170 min only a small additional decrease to 10.6 + 0.5 (3) pg of ascorbic acid/mg of DNA was measured. When lung slices were incubated for 5 min in a shaker, 51.5% of the original tissue content of ascorbic acid and 29.3% of the protein were recovered in the medium (Table II). The proportion of lactate dehydrogenase activity and potassium released was 17.1% and 22% respectively. Sliced lung that had first been perfused with saline via the pulmonary artery, released 17.2% of its protein. About 6% of the original DNA content of lung slices was recovered in the medium irrespective of whether or not the lungs were perfused prior to slicing. Lung slices had an oxygen consumption of 8.68 ± 0.16 (4) pl/mg dry wt./h and an aerobic lactate production of 0.45 -+ 0.04 (4) pg/mg dry wt./h. Incubation of washed slices with media containing 0--320 pg of ascorbic acid/ml, produced m a x i m u m tissue levels of ascorbic acid within 2 min, after which little change occurred in the next 28 min. Equilibrium values in this experiment show a linear relation between the medium ascorbic acid concentration and the tissue ascorbic acid content (Fig. 2). Centrifugation of homogenized washed lung slices produced supernatants that contained 84.0 ± 3.7 (5) % of the total (supernatant + pellet) ascorbic acid, 4.4 t 1.6 (4) % of the total DNA, and 52.3 + 5.7 (4) % of the protein. After chromatography on G-25 Sephadex, a clear separation of the protein and TABLE
I
THE PROPORTION OF THE ORIGINAL ASCORBIC LUNG SLICES THAT IS REMOVED BY WASHING V a l u e s a r e m e a n s +-S.E. w i t h t h e n u m b e r
Fresh lung Washed lung R e c o v e r e d in w a s h
of observations
ACID,
DNA,
AND
PROTEIN
shown in parenthesis.
Ascorbic acid (~zg/mg D N A )
DNA (% of original)
Protein (% o f o r i g i n a l )
2 2 . 7 + 1.1 ( 5 ) 10.0 ± 0.6 (5) 12.1 + 0.5 (4)
-9 2 . 7 + 1.9 ( 4 ) 7.3 + 1.9 (4)
-72.9 ± 3.7 (4) 27.1 ± 3.7 (4)
CONTENT
OF
112
T A B L E II THE RELEASE OF TISSUE CONSTITUENTS UM AFTER 5 MIN
FROM
LUNG SLICES INTO THE INCUBATION
MEDI-
V a l u e s are m e a n s o f f o u r o b s e r v a t i o n s -+S.E. T h e r e s u l t s axe f o r s l i c e s o f f r e s h l y e x c i s e d l u n g . ' P e r f u s e d lung' means that the intact lung was perfused via the pulmonary artery before slicing. Tissue constituent
Percent of the initial tissue content f o u n d in the m e d i u m
Ascorbic acid Protein Lactate dehydrogenase activity Potassium Protein (perfused lung) DNA DNA (perfused lung)
51.5 29.3 17.1 22.0 17.2 6.8 6.3
+ 2.2 + 2.3 -+ 0 . 6 -+ 2 . 0 + 1.9 + 0.8 + 0.6
both the reduced and oxidized ascorbic acid was obtained (Fig. 3). Incubation of washed lung slices with fluoride, cyanide, or fluoride and cyanide together, produced decreases in the tissue ascorbic acid content of 19%, 28%, and 44% respectively (Table III). In this experiment, ascorbic acid that was lost from the tissue could be recovered in the medium. The inulin space was estimated to be 0.20 + 0.03 (6) ml/g wet weight of tissue and the plasma ascorbic acid concentration was measured to be 14.0 _+ 1.6 (9) pg/ml.
Guinea pig experiments Guinea pigs fed the ascorbic acid-deficient diet (supp. 0) lost weight after about the twelfth day and died approximately 10 days later, showing acute 30
~-
20
20'/0 I n u l i n
?g
space
o
161 i-=
I
I
I
100 200 300 Medium ascorbic acid ~' (pg/ml)
i
400
F i g . 2. A s c o r b i c a c i d c o n t e n t o f t i s s u e a n d m e d i u m a t e q u i l i b r i u m . V a l u e s are m e a n s w i t h t h e n u m b e r o f o b s e r v a t i o n s s h o w n i n p a r e n t h e s i s . T h e d o t size c o v e r s -+S.E. T h e r a p i d i t y w i t h w h i c h a s c o r b i c a c i d c a n a c c u m u l a t e i n t i s s u e w h e n s l i c e s are i n c u b a t e d w i t h m e d i a c o n t a i n i n g a s c o r b i c a c i d s u g g e s t s t h a t i t is t h e i n t e r s t i t i a l f l u i d s p a c e t h a t is f i l l e d w i t h a s e o r b i c a c i d . H o w e v e r , s o m e d i f f u s i o n i n t o t h e t i s s u e c o m p a r t ment must have occurred since calculation based on an inulin space of 0.2 ml/g of tissue shows that only p a r t o f t h e tissue levels of a s c o r b i c acid at e q u i l i b r i u m can be a c c o u n t e d for on this basis.
113
15
15
.-& 10 E
~ ~ L o_
10 ~ c) o (3-
Protein
AH2
5 S.
5
i
5
0
15
Fraction
20 25 number
Fig. 3. S e p h a d e x ( G - 2 5 ) s e p a r a t i o n Oxidized ascorbic acid, (A).
30
35
o f an e x t r a c t o f w a s h e d l u n g slices. R e d u c e d a s c o r b i c a c i d , ( A H 2 ) ;
signs of scurvy. At 20 days, the organ ascorbic acid levels were found to be negligable in comparison to those of supp. 10 guinea pigs. In the incubation experiments, 57, 58 and 78% of the original ascorbic acid content of lung slices from supp. 10, supp. 2, and supp. 0 guinea pigs respectively were found in the medium following incubation with Krebs-phosphate solution (Table IV). Slices from supp. 0 guinea pigs released about twice the amount of D N A into the medium than did slices from supp. 10 animals and they released about 9% more protein. Supp. 2 slices lost intermediate quantities of D N A and protein. Discussion Extracellular ascorbic acid in rat lung The rapid removal of about half the ascorbic acid content of lung slices by washing suggests that this ascorbic acid may be extracellular. The loss of whole cells and the contents of damaged cells will contribute some ascorbic acid to
T A B L E III THE EFFECT OF FLUORIDE LUNG SLICES
AND CYANIDE ON THE ASCORBIC ACID CONTENT OF WASHED
V a l u e s are m e a n s o f t h r e e o b s e r v a t i o n s ± S . E . M e a n s w i t h like s u p e r s c r i p t d i f f e r s i g n i f i c a n t l y u s i n g S t u dent's t test with P < 0.05. Treatment
T i s s u e a s c o r b i c acid c o n t e n t (pg/mg DNA)
F r e s h l u n g slices W a s h e d l u n g slices a f t e r i n c u b a t i o n Fluoride (10 raM) C y a n i d e (2 r a M ) Fluoride and cyanide
22.98 10.27 8.35 7.43 5.78
+ ~ * ± ±
0.33 0.17 0.59 0.22 0.20
a abcd b c d
114 T A B L E IV THE DISTRIBUTION OF ASCORBIC ACID, DNA, AND PROTEIN BETWEEN THE TISSUE AND MEDIUM AFTER INCUBATION OF LUNG SLICES FROM ASCORBIC ACID-SUPPLEMENTED AND ASCORBIC ACID-DEFICIENT GUINEA PIGS IN KREBS-PHOSPHATE FOR 1 h V a l u e s are m e a n s +-S.E. w i t h t h e n u m b e r o f o b s e r v a t i o n s s h o w n i n p a r e n t h e s i s . Ascorbic acid supplement (rag/100 g/day)
Tissue ascorbic acid (/zg/mg DNA)
Medium ascorbic acid (pg/mg DNA)
Medium DNA (% o f t o t a l )
Medium protein (% o f t o t a l )
10 2 None
6 . 9 6 ÷ 0 . 5 7 (S) 0.80 + 0.06 (16) 0 . 2 1 ± 0 . 0 4 (5)
9 . 2 8 ÷ 0 . 9 0 (8) 1.09 ~ 0.09 (16) 0 . 6 3 ~ 0 . 0 3 (5)
9.0 t 0.9 (8) 12.1 + 1.3 ( 1 6 ) 2 0 . 1 4 1.6 ( 7 )
3 5 . 7 " 1.9 (8) 4 0 . 3 ~ 1.3 ( 1 6 ) 4 4 . 6 + 0 . 6 (7)
the total a m o u n t washed out of the slices, but the size of this cont ri but i on is difficult to estimate. When slices are incubated for 5 min, about half of the original tissue ascorbic acid c o n t e n t can be accounted for in the medium and this agrees with the p r o p o r t i o n of ascorbic acid that is removed by washing. 7% o f the original tissue DNA is removed by either washing or incubating the slices and this probably represents the removal of free cells such as alveolar macrophages. Since lung slices were f o u n d to consume oxygen at a rate similar to those described previously for lung [10], and aerobic glycolysis was found to be a quarter of comparable values r e por t ed elsewhere [11], it was considered t h at only a small a m o u n t of cellular damage was caused by the present m e t h o d of slicing. Some of the relatively large a m o u n t of tissue protein that is removed by washing or incubation will be associated with the removal of cells and damaged cell contents, but a p r o p o r t i o n must have also been extracellular and present in the interstitial fluid space, the circulation, or the airspaces. The co n tri but i on of protein from the blood can be seen by the fact t h a t when tissue slices from the perfused lung were incubated, only 17% of the protein was f o u nd in the medium. A similar percentage of the activity of the cytoplasmic en zym e , lactate dehydrogenase, also appeared in the medium. Ano t h er estimate o f cell damage is that 22% of the tissue potassium is found in the medium when slices are incubated. Part of this potassium would be associated with e r y t h r o c y t e s and some would have been extracellular. The potassium co n cen tr atio n in the respiratory tract fluid of man is 13 mM [12] and it m a y be higher in rat since in the submaxillary-sublingual gland secretions of this species, its concentration is about 50 mM [13]. From these results, it is concluded that the e x t e n t of cell loss and damage in the lung slices is about 20%. Th e rapid depletion of an intracellular c o m p a r t m e n t of ascorbic acid by diffusion might also account for the removal of some tissue ascorbic acid by washing, but this is unlikely because the original tissue c o n t e n t of ascorbic acid could n o t be reconstituted by incubating washed tissue slices in media that contained ascorbic acid in excess of the plasma concentration. Th e ready removal of about half the ascorbic acid, c o n t e n t of lung slices by either washing or incubation procedures, taken in conjunction with an estimate o f 20% for the tissue damage caused by slicing, suggests that about 30% of the ascorbic acid in lung is extracellular. Almost none of this ascorbic acid is present in the interstitial fluid space because the plasma concent rat i on of ascorbic
115 acid is 14 pg/ml and the inulin space is 0.2 ml/g of tissue. The present findings agree with the result obtained previously that 30% of the ascorbic acid content of an intact lung can be removed by washing the airspaces [2], and supports the conclusion that ascorbic acid is concentrated in the fluid lining the respiratory epithelium. hztracellular ascorbic acid in rat lung After removal of the extracellular ascorbic acid by washing, the remainder is unaffected by further washing. This residual ascorbic acid may be contained in an actively concentrating compartment since incubation of washed slices with metabolic poisons decreased the tissue content of ascorbic acid and under the mild conditions of Sephadex chromatography, the ascorbic acid in washed lung slices was not found to be protein bound. In adrenal and liver tissue there is evidence that ascorbic acid is b o u n d to protein [14--16], although this has been questioned by Lewis et al. [17]. March and Tolbert [18] have found only non-specific binding of ascorbic acid in guinea pig brain homogenates. In the present experiments, no evidence of protein-bound ascorbic acid has been found in rat lung homogenates under the conditions of sephadex chromatography. Fig. 4 summarizes the characteristics of the ascorbic acid compartments in rat lung and shows how these compartments may interact. Extracellular ascorbic acid in guinea pig lung Lung slices from guinea pigs fed ascorbic acid (supp. 10 and supp. 2) released a b o u t 57% of their ascorbic acid content on incubation. Assuming the same degree of damage as in the rat experiments, about 37% of this ascorbic acid may be extracellular. In ascorbic acid-deficient guinea pigs, where the lung ascorbic acid content drops to about 1% of the original level, 78% of the ascorbic acid is readily removed by incubation. Of this 58% may be extracellular.
Plasma (pglm[)
;sue a/g}
Airspace
Airspace
fluid
(pg/mt)
14
g2
3602100
Fig. 4. P u l m o n a r y ascorbic acid c o m p a r m e n t s . Ascorbic acid f r o m the plasma is transported into a lung tissue c o m p a r t m e n t w h e r e it is concentrated. It is then m o v e d across the respiratory epithelium where it accumulates in the fluid lining the airspaces. Calculation of the values in the figure is based o n a lung ascorbic acid content of 2 4 0 pg/g J21 J of which 7 0 % is tissue ascorbic and 3 0 % is extracellular. It is assumed t h a t t h e airspace fluid h a s a v o l u m e o f f r o m 0 . 0 3 m l / g J2] t o 0 . 2 m l / g .
116
Although some of the extracellular ascorbic acid in the incubation media will be derived from oxidized ascorbic acid that is reduced by glutathione, it is suggested that the function of extracellular ascorbic acid in guinea pig lung is of sufficient importance for this ascorbic acid c o m p a r t m e n t to be maintained in p r o p o r t i o n to the intracellular pool of ascorbic acid, even in acute scurvy. Guinea pig (supp. 10) lung slices, like rat lung slices, release about 30% of their total protein and a bout 8% of their total DNA when t hey are incubated. However, when lung slices from suppl. 2 and suppl 0 guinea pigs are incubated there is an increase in the a m o u n t of protein and DNA released into the medium which is directly proportional to the deg-fee of ascorbic acid deficiency. A likely explanation is that since ascorbic acid is i m p o r t a n t in maintaining the intercellular matrix, it might be expect ed that cells will shake loose from ascorbic acid-deficient tissue slices on incubation. The increase in the a m o u n t of protein in the medium would thus be associated with the extra DNA released. A s c o r b i c acid as an extracellular a n t i o x i d a n t in lung
Since the lung is exposed to most of the atmospheric oxygen tension, it is attractive to propose that ascorbic acid might act as an antioxidant in this organ. In experiments where rats were exposed to hyperbaric oxygen [19] or ozone [20] administration of ascorbic acid prior to t r e a t m e n t lessened the toxic effects. Also, exposure to hyperbaric oxygen decreased rat lung ascorbic acid by 40% [21]. Since a b o u t 30% of the ascorbic acid c o n t e n t of rat lung appears to be extracellular and concent r a t e d in the fluid lining the airspaces, it may be proposed that it acts as an antioxidant at this site. Certainly there would seem to be a use for an extraceUular antioxidant in lung. Klaus et al. [22] showed th at the surface activity of an extract of surfactant from beef lung was gradually lost over 2 h if exposed to air. T hey concluded that for surfactant to resist oxygen at the air-tissue interface, it required the antioxidant potential o f substances as ye t unidentified. Also, exposure of snail mucus to o z o n e decreased the inhibition titer of the mucus with the hemagglutination test on the influenza viruses PR-8 and LEE [23]. It is concluded that a b o u t 30% o f the ascorbic acid c o n t e n t of rat and guinea pig lung may be acting as an extracellular antioxidant, protecting some p r o p e r t y of either surfactant or mucus. References 1 2 3 4 5 6 7 8 9 10 11 12 13
Willis, R . J . and Kratzing, C.C. ( 1 9 7 5 ) P f l u g e r s A r c h . 3 5 6 , 9 3 - - 9 8 Willis, R . J . and Kratzing, C . C . ( 1 9 7 4 ) B i o c h e m . B i o p h y s . R e s . C o m m u n . 5 9 , 1 2 5 0 - - 1 2 5 3 B o l i n , D.W. a n d B o o k , L. ( 1 9 4 7 ) S c i e n c e 1 0 6 , 4 5 1 Burton, K. (1956) Biochem. J. 62,315--323 G o a , J. ( 1 9 5 3 ) S c a n d . J . Clin. L a b . I n v e s t . 5 , 2 1 8 - - 2 2 2 W r 6 b l e w s k i , I. and Mason, H . S . ( 1 9 5 9 ) P r o c . S o c . E x p . Biol. M e d . 9 0 , 2 1 0 - - 2 1 3 U m b r e i t , W.W., B u r r i s , R . H . a n d S t a u f f e r , J . F . ( 1 9 6 4 ) M a n o m e t r i c T e c h n i q u e s , B u r g e s s P u b l i s h i n g C o . , Minnesota B a r k e r , S.B. a n d S u m m e r s o n , W . H . ( 1 9 4 1 ) J. Biol. C h e m . 1 3 8 , 5 3 5 - - 5 5 4 K u l k a , R . G . ( 1 9 5 6 ) B i o c h e m . J. 6 3 , 5 4 2 - - 5 4 8 B a r t o n , E . S . G . , Miller, Z.B. and Bartlett, G . R . ( 1 9 4 7 ) J . Biol. C h e m . 1 7 1 , 7 9 1 - - 8 0 0 L e v y , S.E. a n d H a r v e y , E. ( 1 9 7 4 ) J . A p p l . P h y s i o l . 3 7 , 2 3 9 - - 2 4 0 P o t t e r , J . L . , M a t h e w s , L . W . , L e m n , J . a n d S p e c t o r , S. ( 1 9 6 3 ) A n n . N . Y . A c a d . Sci. 1 0 6 , 6 9 2 - - - 6 9 7 S c h n e y e r , C . A . a n d S c h n e y e r , L . H . ( 1 9 5 9 ) P r o c . Soc. E x p . Biol. M e d . 1 0 1 , 5 6 8 - - 5 6 9
117 14 15 16 17 18 19 20 21 22 23
SumerwlU, W.N. and Sealock, R.R. (1952) J. Biol. Chem. 1 9 6 , 7 5 3 - - 7 5 9 Roe, J.H. and Itscoitz, S.B. (1963) Proc. Soc. Exp. Biol. Med. 1 1 3 , 6 4 6 - - 6 5 0 Fiddick, R. and Heath, H. (1967) Biochim. Biophys. Acta 1 3 6 , 2 0 6 - - 2 1 3 Lewis, W.H., Chiang, J.L. and Gross, S. (1960) Archs. Biochem. Biophys. 89, 21--26 March, S.C. and Tolbert, B.M. (1971) Fed. Proc. 30, 521 Jamieson, D. and van den Brenk, H.A.S. (1964) Biochem. Pharmac. 1 3 , 1 5 9 - - 1 6 4 Matzen, R.N. (1957) J. Appl. Physiol. 1 1 , 1 0 5 - - 1 0 9 Willis, R.J. and Kratzing, C.C. (1972) Am. J. Physiol. 222, 1391--1394 Klaus, M.H., Clements, J.A. and Havel, R.J. (1961) Proc. Natl. Acad. Sci. U.S. 47, 1858--1859 Falk, H.L., Kotin, P. and R o w l e t t e , W. (1963) Ann. N.Y. Aead. Sci. 106, 583--608
