Lung (1983) 161:99-109

Ascorbic Acid in Bronchoalveolar Wash Lorant Skoza. Arthur Snyder and Yutaka Kikkawa* Department of Pathology, New York Medical College, Valhalla. New York 10595, USA

Abstract. The ascorbic acid concentration of bronchoalveolar wash has not been measured directly in the past. When attempts were made to measure this vitamin in the wash. it was found that the existing methods were not sensitive enough to measure the ascorbic acid in these highly dilute samples. Using ascorbic acid standards at concentrations approximating those of sequential wash samples, we have developed an accurate microassay method. The major feature of this new method is reduction of sample dilution by the reagents with the use of trichloroacetic acid in lieu of H2SO~. Using this method, we measured ascorbic acid in the first to the sixth sequential bronchoalveolar wash. the values ranging from about 5.0 ~g/ml to 0.3 p.g/ml. Pooled lavage content of ascorbic acid constituted approximately 10-12% of the whole l u n g ascorbate. The close agreement between the content of reduced ascorbic acid and the total ascorbic acid in the bronchoalveolar lavages establishes that the ascorbic acid in the airway is present in the reduced state. Key words: Lung ascorbic acid - Lung extracellular ascorbic acid - Alveolar lining layer (ALL) - Bronchoalveolar wash.

Introduction

Recent experiments suggest that a portion of the ascorbic acid in the lung may be present in the aqueous layer of alveoli and airways (ALAA). Willis et al. 111 [22-25] used an indirect method to measure the ascorbic acid content of ALAA. Ascorbic acid was measured in the perfused lung [23], in the perfused lung after lavage [23], and in saline extracted samples of lung tissue slices [25]. The authors extrapolated the ascorbic acid content of the ALAA to be between 30-50% of the total lung ascorbic acid [25]. The role of airway ascorbic acid is presently unknown, but it may play an important role in protection of lung cells against oxygen and ozone toxicity [4, 5, *

To whom offprint requests should be sent

100

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12, 14] and instilled chemicals [17]. Direct determination of the ascorbic acid content of the aqueous layer of the airway and alveoli obtained by sequential saline washing is difficult because the recovered samples are diluted over 1500-2000 fold. Accurate measurement of ascorbic acid in the ALAA during oxidant stress might clarify the extracellular role of this vitamin [5]. In this investigation, modified dinitrophenylhydrazine and 2,6-dichlorophenolindophenol assay methods were developed to study the ascorbic acid content of the washes directly. We report here the first direct determination of ascorbic acid content and the redox state of this ascorbic acid in the extracellular aqueous layer of the atveoli and airways.

Materials and Methods

Animals Male, Sprague Dawley, specific pathogen free rats (Microbiological Associates, Walkersville, Md.) weighing 240-370 g were housed 4 per box in hanging wire basket cages and given Purina cat chow and tap water adlibitum. Rats were sacrificed with an overdose of IV sodium pentobarbitol (Abbott Laboratories, N. Chicago, Ill.. 15-20 rag).

Bronchoalveolar Washing The trachea was incised below the thyroid and cannulated. The lavage media (unbuffered saline) was introduced passively from a reservoir at approximately 45 cm height through a tracheal catheter. After a measured volume was introduced, the instilled fluid was withdrawn gently and evenly. When multiple sequential washes were performed, an initial volume of 7 ml was introduced in the first wash to fill the dead space of tubing (approx. 1 ml), thereafter volumes of 6 ml were instilled. In some cases, lavages were performed after the lungs were perfused at pressure of 20 cm H20 with saline via the portal vein. Volume recovery of instilled saline was better than 93% in all experiments (35.1_+0.6 ml, n = 6, mean_+ SD).

Sample Preparation Washes were rendered cell free by centrifugation at 4 0 0 x g for 10 rain. Small amounts of floating foam were removed and the supernatant fluid collected and used as the cell free lavage sample. Cell pellets were used as a source of alveolar macrophages. The pellets were resuspended in saline, repelleted and homogenized in 1:0 ml of 5% TCA in an Econogrind homogenizer (Radnoti Glass Yech., Inc., Acardia, Cal.). The homogenates were centrifuged at 25,500xg for 20 rain and the supernatant was used as the macrophage fraction. For the measurement of the ascorbic acid content of the whole lung, the lung was dissected free of trachea and large bronchi. The tissue was minced into 1-2 mm cubes in 15 ml of 5% TCA, homogenized in a motor driven Potter-Elvehjem glass-teflon homogenizer (Palo Lab. Supplies, Inc., N.Y.C.). The homogenate was centrifuged at 25,500xg for 20 rain, a foam layer removed by suction and the clear supernatant collected for the analyses.

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Reagents Ascorbic acid standard (Fisher Scientific Co., Fairlawn, N.J.) 100 gg per ml of 0.6% HPO3, and 2.6 dichlorophenolindophenol dye-stock (DCIP dye, Sigma Chemical Co., St. Louis. Mo.) 0.1% in distilled water were stored at 4 ° C. Thiourea Stock (Eastman Organic Chemicals, Rochester, N.Y.) was prepared as 10% solution in 50% ethanol (w/v). Prior to use the stock solution was diluted 1 to 5 with distilled water, p-Chloromercuribenzoic Acid (pCMB, Sigma Chemical Co., St. Louis, Mo.) was prepared as 0.3% solution in 0.05 N NaOH. Roe's DNPH reagent (Eastman Organic Chemicals. Rochester, N.Y.) was prepared with 2% DNPH and 4% thiourea in 9 N H~SO4 [18. 19. 20]. Bessey's C u / D N P H reagent was prepared with 2% DNPH. 0.25% thiourea. 0.03% CuSO4 • 5H20 in 9 N HaSO4 [2, 11].

DNPH Assays (Oxidized A scorhic Acid Assay) Deproteinization and stabilization of the ascorbic acid in the acellular wash were accomplished by addition of 0.125 ml 6.0% HPO3 per ml of wash and centrifugation at 23,500x g for 15 rain. The alliquots of clear supernatant fluid were used in the two ascorbic acid determinations described below.

Method A. Simultaneous oxidation and coupling of ascorbic acid. 0.10ml of C u / D N P H was added to duplicate 0.6 ml aliquots of wash samples, reagent blanks and reference solutions. The tubes were covered and incubated at 25 ° C in the dark for 24 h. 0.25 ml of 100% TCA was added to each tube (final volume 0.95 ml), the samples mixed and read at 520 nm.

Method B. DCIP oxidation of ascorbic acid prior to the DNPH reaction. 0.015 ml of DCIP stock solution was added to duplicate 0.6 ml aliquots of deproteinized samples, reagent blanks and reference solutions. After 2 rain the excess dye was decolorized by the addition of 0.015 mI of the thiourea stock solution, 0.12 ml of Roe's DNPH reagent was then added to each tube. The samples were incubated at 25°C in the dark for 24 h followed by the addition of 0.25 ml of 100% TCA to each tube (final volume 1.0 ml). Samples were mixed and read at 520 rim. The ascorbic acid content for both assays A and B was calculated by a modified method of Henry [7]. Correction was made for sample turbidity by reading samples at 600 nm. The range of the above assays was 0.25-5.0 gg ascorbic acid per sample. Reduced A scorbic Acid Assay The processing procedure of the wash fluid was modified in order to stabilize ascorbic acid as quickly as possible after removaI from the lungs. Wash fluid was filtered with a 0.22 ~m Millipore filter (Swinex, Millipore Corp., Bedford, Mass.) to remove the cells. The filtrate was deproteinized by addition of 0.125 ml 6.0% HPO3 per ml of wash and was centrifuged at 23,500× g for 15 rain. A foamy top layer was removed by suction and the supernatant fluid was treated with 0.3 ml of stock p-chloromercuribenzoic acid (pCMB) solution and centrifuged for 15 min at 23,500xg to remove the precipitate [161. Omission of pCMB treatment of wash fluid leads to a 13-15% overestimate of reduced ascorbic acid concentration.

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I I 1 I I 0 I0 20 30 40 50 F I N A L C O N C E N T R A T I O N OF C O L O R D E V E L O P E R (%)

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Fig. 1. Relationship between the molar extinction coefficient of the ascorbic acid derivative and the concentration of TCA or H~SO,. After DCIP oxidation and reaction with DNPH the color was developed by addition of increasing amounts of 100% TCA (open circles) or by 85% H2SO4 (closed circles). Molar extinction coefficient was calculated from the absorbance at 520 nm of samples. Arrow No. 1 shows the limit of sensitivity using Roe's method (42.5% H2SO4), while Arrow No. 2 is that of Bessey's (36% H2SO4). Arrow No. 3 indicates the increased sensitivity using TCA (25%)

Duplicate 1.0 ml aliquots of HPO3 deproteinized, pCMB treated wash samples, containing 2.0-7.0 gg ascorbic acid per ml, were placed in test tubes. Controls included reference solutions. To all tubes 0.05 ml of 12.5% Na citrate was added to adjust ~he pH to 3.5 + 0.5. To these buffered samples, 0.02 ml of stock D C I P dye (0.1% in distilled water) was added, mixed, and each sample individually read within 30 s at 520 nm against a 1.5% HPO3 blank. Sample and dye control background readings were obtained by the addition of ascorbic acid to decolorize excess DCIP dye. Calculation of reduced ascorbic acid levels were performed as described by Henry [7, 81. Results and Discussion

Measurements of Reference A scorbic Acid The principal steps o f D N P H assays are: 1. The oxidation ofascorbic acid. 2. Coupling of the oxidized ascorbic acid with D P N H reagents. The product of the reaction is insoluble osazone. 3. Color development by the addition of strong acid which solubilizes the osazone [18]. The measurement of ascorbic acid in lavage samples required the development o f a microassay technique. The most significant dilution o f the sample is by the 1 : 1 (v/v) or more addition of concentrated H2SO4 used as a color developer [2, 3, 7, 9, 15, 18, 19]. Figure 1 shows the experiments with decreasing concentrations of H2SO4, which was added to the tubes containing osazones of 5 ~tg DCIP oxidized

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Fig. 2. Effect of DNPH coupling conditions and color developers on ascorbic acid calibration curves. The volume of each DNPH reaction mixture was 0.75 ml. Line 1. DNPH reaction: 37°C, 3 h, 857o H~SO4 [20]. Best fit line: y=-0.008+0.085 X; correlation coefficient r=0.998. Line 2. DNPH reaction: 37°C, 3 h, 1007o TCA (method A). Best fit line: y = -0.0125 +0.127 X; r=0.994. Line 3. Primary DNPH reaction: 37 ° C, 3 h: secondary DNPH reaction overnight at room temperature (method A). Best fit line: y=0.0001+0.165X; r=0.999. Note the increasing sensitivity that can be achieved by the secondary DNPH reaction (line 3)

ascorbic acid standard. The results are expressed as the relationship between molar extinction coefficient (~) and the final concentration of H2SO~ in the reaction mixture (closed circles). Arrow No. 1 shows the limit o f sensitivity using Roe's method, while Arrow No. 2 is that o f Bessey's. The concentration can be lowered to 31.5% before the ~ value falls. These results suggest that there is little i m p r o v e m e n t with the use u f H z S Q in more dilute ascorbic acid samples. According to Roe, the basic principle o f color d e v e l o p m e n t in the D N P H assay is to dehydrate the osazone derivatives with strong acid such as with 85% H2SO4 [13]. It was deemed possible to use an even stronger acid for the s a m e purpose. Therefore, 100% T C A solution was used as a color developer. The advantage o f T C A is clearly illustrated in Fig. 1, because less volume was n e e d e d to obtain a constant molar extinction coefficient (~) (open circles). F i n a l concentration o f 15% T C A is all that was required to obtain constant ~. A d d i t i o n o f T C A b e y o n d these minimal volumes, up to 75% final concentration did not p r o d u c e a n y adverse effects on the samples. To insure constant ~ we have a d o p t e d 25% T C A as the final concentration (Arrow 3, Fig. 1). The effect o f the two color developers on the sensitivity o f otherwise similar D N P H assays can be seen in Fig. 2. In these experiments, a k n o w n quantity o f

L. Skoza et al.

104 0,5

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Fig. 3A, B. Effect of 25% TCA on the DNPH reaction of oxidized ascorbic acid. The primary DNPH reaction (dark bars) was carried out with 2 ,ttg ascorbic acid by method A at 37 ° C (A) and by method B at 60° C (B) for varying lengths of time. The secondary DNPH reaction (light bars) was carried out overnight at room temperature standard ascorbic acid was reacted for 3 h with DNPH. Line l indicates the absorbance readings after 85% H2SO4 was used (42.5% final concentration of H2SO4 in samples), while Line 2 shows the reading after 100% T C A (25% final concentration of T C A in samples). The data show that the use of TCA increased the sensitivity of the assay by a factor of 1.5. There are some other advantages in the use of TCA. Addition of HzSO4 to the D N P H reaction mixture stops the osazone formation immediately (data not shown). In contrast, with TCA, the D N P H reaction progresses at an unaltered rate. The absorbance of the samples at 520 nm were the same, regardless of whether T C A was added before or after the D N P H reaction. This property of T C A is shown in Fig. 3. In" these experiments, identical samples containing 2.0 ~tg ascorbic acid were reacted with the D N P H reagents for varying lengths of time. The dark bars in Fig. 3 A and B show the progress of the color reaction at 3 7 ° C (temperature modification of method A) and 60°C (temperature modification of method B). At the time specified in the figures, the reaction was terminated by a quick chilling of the samples followed by the addition of TCA. After recording the absorbance o f the samples at 520 nm (dark bars), they were then further incubated overnight at room temperature. This secondary D N P H reaction is shown by the light bars. T h e results show that under conventional ascorbic acid assays (i.e. 3 - 4 h at 3 7 ° C or 2 - 5 h at 60 ° C) the reaction of D N P H with the oxidized ascorbic acid is far from complete. A secondary D N P H reaction was necessary to achieve an apparent

Ascorbic Acid in Pulmonary Lavage

105

Table 1. Absorbances of ascorbic acid standards after reaction with DNPH at room temperature for 24 h. Ascorbic acid standards were assayed by the Cu2÷/DPNH reagent. Equation of the best fit line was y = - 0.001 + 0.147X; correlation coefficient r = 0.999 ~tg Ascorbic acid per assay [X]

Absorbance at 520 nm [Yl

Molar extinction coefficientb

0.25 0.50 0.75 1.00 2.00 3.00 5.00

0.038 + 0.003 a 0.072 _+0.002 0.111 +0.003 0.143 + 0.002 0.287 +__0.009 0.445 ___0.004 0.732 + 0.014

26.77 25.36 26.07 25.18 25.28 26.13 25.79 25.80+0.57

b

Absorbance of the samples at 520 nm, mean_+_SD (n=6) The molar extinction coefficient was calculated from the mean absorbance of the samples

endpoint at both 37°C and 60 ° C, although the rate o f the reaction at 6 0 ° C was more rapid than at 37 ° C. According to Zloch et al., m the colormetric d e t e r m i n a t i o n o f ascorbic acid by the conventional D N P H assays, only a fraction of the vitamin present was really determined, because the D N P H reaction was still progressing in the exponential phase [26]. The time o f the completed D N P H reaction can be shortened substantially by using elevated reaction temperatures. But the possibility exists that decomposition o f oxidized ascorbic acid m a y occur, a n d osazones o f interfering materials may also form [20]. Our room temperature incubation m i n i m i z e d this interference as shown in Table 1. The constant shows that a strict proportionality exists between the absorbance o f the samples at 520 nm and its respective concentrations. The absorbance o f a sample containing 1.0 ~tg ascorbic acid was 0.143 at 520 nm. This value is quite close to 0.147, a value calculated from equation o f the best fit line, and shows the sensity o f this assay. This can be further increased if the reaction is carried out initially for 3 h at 37 ° C a n d then 17 h at 25 ° C, as shown in Fig. 2, Line 3. The absorbance o f a sample containing a 1.0 ~tg ascorbic acid was 0.165, indicating the extreme sensitivity o f this reaction in s u b m i c r o g r a m range o f concentration.

Ascorbic Acid Content of Bronchoalveolar Washes The pattern of removal of ascorbic acid by sequential saline washes from the airway of a single rat is shown in Fig. 4. The figure shows the ascorbic acid concentration (left scale) and total content (right scale) in each i n d i v i d u a l lavage. A r a p i d decrease in ascorbic acid concentration for each sequential lavage is exhibited. After the third lavage, the concentration o f ascorbic acid fell below 1.0 p,g/ml. By the sixth lavage, the concentration reached the detection limit.

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A scorbic Acid Content ofA lveolar macrophages Analysis of cell pellets required special consideration because the sample size was very limited. The ascorbic acid content of the cell pellets was about 3 ttg per pellet (Table 2). The pellet was extracted with 1 ml of 5% TCA solution. For duplicate analysis of the samples, a proportional reduction of the assay was necessary. To achieve a reaction mixture of 0.5 mL 0.3 ml of the sample, 0.05 ml of C u / D N P H reagent and 0.15 ml of 100% TCA were used. This gave an absorbance for a l~tg ascorbic acid mixture of 0.280-0.300 after 24 h incubation at room temperature.

A scorbic Acid Content of Lung Tissue Table 2 shows the distribution of ascorbic acid in the lung tissue. It can be seen that six sequential saline washes removed about 10-12% of the total ascorbic acid content in the tung tissue from either intact or perfused animals. The cellular pellets of the saline washes contained less than i% of the ascorbic acid. The sum of the ascorbic acid found in the various tung compartments was in reasonably good agreement with the ascorbic acid content of the whole intact lungs.

Redox State of Ascorbic Acid in Bronchoalveolar Wash The in vivo existence of the oxidized forms of ascorbic acid, dehydroascorbic acid (A) and diketogulonic acid (DKG), is uncertain, although they can be formed by oxidation of ascorbic acid in aqueous solution in vitro [16]. In non-pulmonary tissue these oxidized forms are reported to be very low or nonexistent [7, 9, 16, 21]. To shorten the sample handling from 30 min to I rain single saline wash was quickly filtered upon removal from the lung. The filtrate was immediately stabilized and deproteinized by addition of HPOs.

b c

31 t.5 _+56.2 (n = 26) 292.3_+ 52.8 (n = 14) 277.8 +- 31.7 (n = 10)

47,36_ 6.90 p.g 50.86+_ 7.21

Acellular washes °

Samples obtained by six 6.0 ml sequential washes Ascorbic acid remained in the lung tissue after the six saline lavages Mathematical sum of the ascorbic acid content of the lung compartments

Group 1 Group It Group 11I

Weight of animals

3.00 +- 0.81 3.27 +- 0.96

Cell pellets"

429.7 +_55.8 366.5 +_68.0

Lung tissue b

497.96 -t- 57.1 459.8 _+49.9 ~ 420.6 _+71.3 °

Total ascorbic acid content in lung

Table 2. Distribution of ascorbic acid in the lung tissue. Group 1: Ascorbic acid in intact ltmgs. Group 11: Ascorbic acid content of the lung compartments in saline perfused animals. Group III: Ascorbic acid content of tb.e lung compartments in unperfused animals

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108 Table 3. Comparison of reduced and total ascorbic acid

Mean+SD

Reduced ascorbic acid (ag/ml)

Total ascorbic acid reduced and oxidized (gg/ml)

Difference reduced - total (ttg/mt)

4.95 3.35 5.17 3.52 3.64 5.56 3.57 3.84 4.04

4.84 3.95 5.18 3.39 3.50 5.67 3.11 3.46 4.06

0. l 1 - 0.60 -0.01 0.13 0.14 - 0.11 0.46 0.38 - 0.02

4.18+-0.82"

4.13+_0.09

0.053_+.0.307

Table 3 compares the measurement o f total and reduced ascorbic acid in individual lavages from nine rats. The total ascorbic acid concentration o f 4.13 + 0.09 u g / m l was in good agreement with the reduced ascorbic acid concentration of4.18 ± 0.82 p~g/ml. Paired t test showed no significant difference. This indicates that all the ascorbic acid in the airways exists in the reduced state. It is g e n e r a l l y believed that glutathione (GSH) and other sulfhydryl c o m p o u n d s such as cysteine are probably responsible for maintaining vitamin C in the reduced form in vivo [10, 12]. However, we were not able to detect G S H or other sulfhydryl c o m p o u n d s either in reduced or in oxidized form. The absence o f airway G S H leaves unexplained how ascorbic acid is m a i n t a i n e d in the reduced state in the face o f the high oxygen tension encountered in the airways. Semidehydroascorbic acid reductase, an enzyme shown to be present on the luminal surface o f hepatocytes, m a y also be present in the airways and may be responsible for keeping ascorbic acid in the reduced state [6]. Alternatively, an active transport o f oxidized ascorbic acid through lung epithelium m a y maintain the redox state o f extracellular ascorbic acid. Studies are in progress to evaluate these two possible mechanisms for maintenance o f reduced airway ascorbic acid.

Acknowledgement. This work was supported by NIH grant HL 24817. References 1. Baker EM, Hammer DC. Kennedy JE, Tolbert PM (1973) Interference by ascorbate-2sulfate in the dintrophenylhydrazine assay of ascorbic acid. Anal Biochem 55: 641-642 2. Bessey OA, Lowry OH, Brock MJ (1947) The quantitative determination of ascorbic acid in small amounts of white blood cells and ptatetets. J Biol Chem 168:197-205 3. Bolin DW, Book L (1947) Oxidation of ascorbic acid to dehydroascorbic acid. Science 106:45l 4. Clark JM, Lambertsen CJ (1971) Pulmonary oxygen toxicity: a review. Pharmacol Rev 23:37-133 5. Deneke SM, Fanburg BL (1980) Normobaric oxygen toxicity of the lung. N Engl J Med 303:76-86

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6. Goldenberg H (1980) Insulin inhibits NADH-semidehydro ascorbate reductase in rat liver plasma membrane. Biochem Res Comm 94:721-726 7. Henry RJ (1946) Vitamin C. In: Clinical chemistry: principles and techniques, Ist edn. Harper & Row, New York, pp 710-719 8. Henry RJ, Cannon DC, Winkelman JW (1974) Determination of vitamin C. In: Clinical chemistry: principles and techniques. 2nd edn. Harper & Row, New York, pp 1393-1398 9. Iggo B, Owen JA, Stewart CP (1956) The determination of vitamin C in normal human plasma and erythrocytes. Clin Chim Acta I: 167-177 I0. Lewin S (1976) Vitamin C: its molecular biology and medical potential. Academic Press, London New York San Francisco 11. Lowry OH, Lopez JA, Bessey OA (1947) The determination of ascorbic acid in small amounts of blood serum. J Biol Chem 196:609-615 12. Menzel DB: Toxicity of ozone, oxygen and radiation. Ann Rev Pharmacol 10: 379-394 13. Mindlin R, Butler A (1938) The determination of ascorbic acid in plasma: a macromethod and micromethod. J Biol Chem 122:673-686 14. Mustafa MG. Tierny DF (1978) Biochemical and metabolic changes in the lung with oxygen, ozone, and nitrogen dioxide toxicity. Am Rev Respir Dis 118: 106l- 1090 15. Omaye ST, Turnbull JD, Sauberlich HE (1979) Selected methods for the determination of ascorbic acid in animal cells, tissue and fluids. Methods Enzymol 62:3-24 16. Owen J, Iggo B (1956) The use of p-chloromercuribenzoic acid in the determination of ascorbic acid with 2 : 6-dichlorophenolindophenol. Biochem J 62:675-680 17. Penha PD, Werthamer S (1973) The role of Pneumocyte II in alveolar injury and repair. Am Rev Respir Dis 107:1109 18. Roe JH, Kuether CA (1943) The determination of ascorbic acid in whole blood and urine through the 2A-ditrophenylhydrazine derivative of dehydroascorbic acid. J Biol Chem 147:399-407 19. Roe JH (1954) Chemical determination of ascorbic, dehydroascorbic and diketogulonic acids. Methods Biochem Anal 1:I 15-139 20. Roe JH (1961) Appraisal of methods for the determination of L-ascorbic acid. Ann NY Acad Sci 92:277-283 2l. Stewart CP, Horn DP, Robson JS (1953) The effect of cortisone and adrenocorticotropic hormone on the dehydroascorbic acid of human plasma. Biochem J 53:254-261 22. Willis RJ, Kratzing GC (1972) Effect of hyperbaric oxygen and norepinephrine on the level of lung ascorbic acid. Am J Physiol 222:1391-1294 23. Willis RJ, Kratzing GC (1974) Ascorbic acid in rat lung. Biochem Biophys Res Commun 59:1250-1253 24. Willis R J, Kratzing GC (1975) Transport of ascorbic acid in perfused rat lung. PfliJgers Arch 356:93-98 25. Willis R J, Kratzing GC (1976) Extracellular ascorbic acid in lung. Biochim Biophys Acta 444: 108-117 26. Zloch Z, Vervev J, Ginter E (1971) Radiochemical evaluation of the 2,4-dinitrophenylhydrazine method for determination of vitamin C. Anal Biochem 43:99-106 Accepted for publication: 24 May 1982