Kinetic Behavior of Ascorbic Acid in Guinea Pigs - American Chemical

14 Kinetic Behavior of Ascorbic A c i d i n

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 25, 2018 | https://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch014

Guinea Pigs DIETRICH HORNIG and DIETER HARTMANN Department of Vitamin and Nutrition Research and Section for Mathematical Statistics, F . Hoffmann-La Roche & Company, Limited, CH-4002, Basle, Switzerland

The course of carbon-14-radioactivity derived from oral (1- C)ascorbic acid in plasma and several tissues was studied in male guinea pigs up to 320 h after intake. The excretion of label was followed in respiratory carbon diox ide, urine, and feces. The evaluation by pharmacokinetic principles yielded an overall half-life of 61 h and a body pool of 21 mg with a total turnover of about 10 mg/d. The total turnover of ascorbate is lower than the daily intake (16 mg/d), indicating incomplete absorption. Ascor bic acid seemed to be bound in several tissues (adrenals, testes) to a higher percentage than in plasma. The maxi mum rate of excretion as carbon dioxide occurred at 0.5 h, whereas peak concentration of radioactivity in plasma was reached at 1.5 h. Therefore, presystemic metabolism must be considered. 14

In guinea pigs respiratory exhalation of carbon- 14-labeled carbon dioxide after administration of (1- C) ascorbic acid is the major route of catabolism, whereas urinary and fecal excretion of unchanged ascorbic acid and labeled metabolites contribute only to a minor part (1). From experiments in guinea pigs receiving (1- C) ascorbic acid by intraperi toneal injection together with 5 mg ascorbic acid, the half-lives of the label in liver, heart, kidneys, adrenals, and spleen compared very well (2-3 d); in the brain this time period was about 5 d (2). This observa tion confirmed earlier findings on the overall biological half-life of ascorbic acid to be about 4 d in guinea pigs (3). Many reports demonstrating the distribution of ascorbic acid in tissues of guinea pigs have appeared. Because this species is not able to A

14

14

0065-2393/82/0200-0293$06.75/0 © 1982 American Chemical Society Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

294

ASCORBIC

ACID

synthesize a s c o r b i c a c i d , t h e c o n c e n t r a t i o n of a s c o r b i c a c i d is d e p e n d e n t o n i n t a k e . T h e h i g h e s t concentrations w e r e d e t e r m i n e d i n t h e a d r e n a l a n d p i t u i t a r y g l a n d s ; l i v e r , b r a i n , a n d s p l e e n s h o w e d c o m p a r a b l e levels. L e s s a s c o r b i c a c i d w a s f o u n d i n h e a r t a n d s k e l e t a l muscles, b o n e m a r r o w , a n d testes

(4).

T h e d i s t r i b u t i o n of l a b e l e d m a t e r i a l f o l l o w i n g a single o r a l or i n t r a


p e r i t o n e a l a d m i n i s t r a t i o n of

( 1 - C ) a s c o r b i c a c i d has b e e n s t u d i e d i n 14

rats (5,6), m i c e (6), a n d g u i n e a p i g s (7-10) e m p l o y i n g either a tissue d i s s e c t i o n m e t h o d or w h o l e b o d y a u t o r a d i o g r a p h y .

T h e s e investigations

suggested t h a t the v a r i o u s tissues m i g h t b e classified i n t o three

groups

a c c o r d i n g to u p t a k e a n d r e t e n t i o n of l a b e l e d m a t e r i a l : a ) tissues w i t h a l o w r e t e n t i o n c a p a c i t y s u c h as l i v e r , l u n g s , k i d n e y s , spleen, b o n e m a r r o w , and lacrimal and parotid glands; b )

tissues e x h i b i t i n g a h i g h r e t e n t i o n

c a p a c i t y a n d also a c o m p a r a b l e h i g h a n d r a p i d a c c u m u l a t i o n of r a d i o a c t i v i t y s u c h as a d r e n a l a n d p i t u i t a r y g l a n d s , s u b m a n d i b u l a r g l a n d s , a n d p a n c r e a s ; a n d c ) tissues h a v i n g a v e r y s t r o n g r e t e n t i o n c a p a c i t y b u t w i t h a l o n g - l a s t i n g , c o n t i n u i n g u p t a k e of l a b e l e d m a t e r i a l . T o the last g r o u p b e l o n g c e r e b r u m , c e r e b e l l u m , b u l b u s o l f a c t o r i u s , a n d testes ( 7 , 9 , 1 0 ) . T o a c h i e v e a m o r e p r e c i s e k n o w l e d g e of the fate of a s c o r b i c a c i d w e h a v e i n v e s t i g a t e d i n g u i n e a p i g s the k i n e t i c b e h a v i o r of the c a r bon-14-label dose.

derived from

( 1 - C ) a s c o r b i c a c i d g i v e n as a single o r a l 14

I n p a r t i c u l a r , the d i s t r i b u t i o n of r a d i o a c t i v i t y i n v a r i o u s tissues

(tissue b i n d i n g a n d t u r n o v e r ) a n d of e l i m i n a t i o n ( r e s p i r a t i o n , u r i n a r y a n d fecal excretion)

was a i m e d at. T h e o b t a i n e d d a t a w e r e

evaluated

using pharmacokinetic principles.

Materials

and Methods

Animals. Male guinea pigs (Himalayan spotted) were maintained prior to the experiment on a vitamin-C-free diet (Nutritional Biochemical Corpora tion) supplemented with 0.5 g of ascorbic acid/kg of diet and fortified with all other vitamins (II). The initial weight was 309 ± 22 (g ± sd), at the time of sacrifice of the animals 327 ± 37 (g ± sd). During the experiment the animals had free access to water and diet. Altogether twenty-eight animals were taken into the experiment. At each time point one animal was killed. Radioacitvity and Dosage. L - ( 1- C)Ascorbic acid (New England Nu clear, NEC-146, spec. act. 8.44 mCi/mmol, purity at least 98.0% ) was dissolved in water and given orally to each animal (50 / / C i ; 250 fxL; 1.04 mg). Respiratory Carbon Dioxide; Urine and Feces. After dosage of the labeled ascorbic acid, the animal was immediately placed in a closed, tight metabolic chamber equipped with a trap for the collection of urine and feces. The incoming air passed with a flow of 280 L / h through the metabolic chamber (volume ~ 22 L ) by suction from an electric pump. The respiratory carbon dioxide was absorbed by ethanol amine (Merck) contained in two flasks (50 mL) that were connected to the chamber by tubes. To determine the absorbed carbon dioxide, the two flasks could be removed after the outcoming airstream 14

Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


14.

295

Kinetic Behavior of Ascorbic Acid

HORNIG AND H A R T M A N N

had been turned around into the new flasks. T h e samples were taken at pre determined time intervals. W i t h this system, it was possible to change the flasks at any time without stopping the flow longer than a few seconds. F r o m each w i t h d r a w n flask, an aliquot of 500 /xL was added to a mixture of 10 m L of ethanol and 10 m L of scintillator solution [0.8% butyl-1,3,4-phenylbiphenylyloxadiazole ( C i b a ) i n toluene]. D u r i n g the experiment, urine and feces were collected every 24 h . Dissection of Tissues. Guinea pigs were anaesthetized w i t h Penthrane (Abbott Laboratories, Incorporated) at predetermined time intervals (15, 30, 45, and 90 m i n ; 2, 4, 6, 8, and 12 h ; 1, 1.5, 2, 2.5, 3, 4, 5, 7, 9, 10, 11, 12, a n d 13d). The abdomen was opened and the animals were sacrificed by bleeding caused by a cut through the vena cava. The blood was collected for determina tion of radioactivity. After centrifugation radioactivity and ascorbic acid c o n centration i n plasma were determined. The tissues were dissected rapidly and kept frozen until processing for determination of radioactivity and ascorbic acid concentration. Determination of Radioactivity. A l l samples were counted i n a Nuclear Chicago Isocap 300 l i q u i d scintillation counter equipped w i t h a Teletype com puterized for direct calculation of disintegrations per minute. F i f t y microliters of blood were directly counted for radioactivity after solubilization (1 m L of 1 N N a O H ) . After incubation at room temperature for 15 m i n the sample was decolorized by adding 200 / i L of hydrogen peroxide and incubating at 80° C for 30 m i n . The processed samples were mixed w i t h 100 / / L of 8 0 % acetic acid and 15 m L of Insta-gel ( P a c k a r d ) , and were counted. Approximately 6 0 - 1 0 0 m g of tissue were solubilized following the same method as for blood. F r o m the collected urine an aliquot (2 m L ) was counted directly w i t h 15 m L Insta-gel. The feces were dried overnight at room temperature, and a 6 0 - 1 0 0 m g aliquot was combusted (12) and counted for radioactivity. Determination of Ascorbic A c i d . w i t h 4.5 m L of 5 % metaphosphoric analyzer using a modified fluorometric tissues was determined according to a

A 500-/xL aliquot of plasma was mixed acid followed by analysis i n an automethod (13). Ascorbic acid content i n published method ( 1 4 ) .

Results Animals.

F r o m the food i n t a k e / 2 4 h a n d an assumed 1 0 0 %

bio

a v a i l a b i l i t y of t h e a s c o r b i c a c i d present i n t h e diet, a t u r n o v e r of 16

±

3 m g a s c o r b i c a c i d / d was c a l c u l a t e d . Radioactivity. of

1 4

T h e specific a c t i v i t y i n p l a s m a , d e f i n e d as 1 0

C - r a d i o a c t i v i t y / m g of u n l a b e l e d a s c o r b i c

d i s s e c t i o n t i m e i n F i g u r e 1.

a c i d , is p l o t t e d

T h e t i m e courses of

1 4

dpm

6

against

C - r a d i o a c t i v i t y for

v a r i o u s tissues are p r e s e n t e d i n F i g u r e s 2—4. The cumulative

1 4

C - r a d i o a c t i v i t y excreted w i t h r e s p i r a t o r y

carbon

d i o x i d e u p to 220 h is s h o w n i n F i g u r e 5. I n F i g u r e 6 the rate of tion

(10

6

dpm/h)

excre

f o r the t i m e p e r i o d u p t o 30 h is p r e s e n t e d ;

the

p e a k appears after a b o u t 0.5 h . T h e c u m u l a t i v e a m o u n t of r a d i o a c t i v i t y ( 1 0

6

dpm)

excreted

with

u r i n e is p l o t t e d i n F i g u r e 7; w h i c h also presents a p l o t of t h e a c c u m u l a t e d radioactivity (10

6

dpm)

e x c r e t e d w i t h t h e feces as a f u n c t i o n of t i m e .


Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982. 6

Time (h)

Figure 1. Specific activity (10 dpm/mg ascorbic acid) vs. time. Circles represent the experimental data. The curve was calculated on the basis of a three-compartment system with first-order absorption.

15-.




14.


297

Kidneys

300

Time (h) Figure 2. Time course of total radioactivity (dpm X 10 ) derived from (l- C)ascorbic acid in liver and kidneys. Continued on next page. 6

14


ASCORBIC

298

ACID


3-1

Time (h) Figure 2 continued. Time course of total radioactivity (dpm X 10 ) derived from (l- C)ascorbic acid in lungs and heart muscle. 6

14


14.

HORNIG

A N D

H A R T M A N N


299

1.0-1


E a

T3 >

0.5

o 03 O "O

co DC

I

1

100

200

300

T i m e (h) Figure 3.

Time course of total radioactivity (dpm X 10 ) derived from (l- C)ascorbic acid in adrenal glands. 6

14

0.05

-i

CD

o X

E a

"O

> 0.025-

•

T i m e (h) Figure 3 continued. Time course of total radioactivity (dpm X 10 ) derived from (l- C)ascorbic acid in putuitary glands. Continued on next page. 6

14


300

ASCORBIC

ACID

10n


o X E a

"O

Time (h) Figure 3 continued. Time course of total radioactivity (dpm X 10 ) derived from (l- C)ascorbic acid in testes. 6

14

1.0

£ x E

-i

0.75-

Time (h) Figure 4.

Time course of total radioactivity (dpm X 10 ) derived from (l- C)ascorbic acid in cerebrum. 6

14


14.

HORNIG

A N D


H A R T M A N N

301

0.3 -|


o

Time (h) Figure 4 continued. Time course of total radioactivity (dmp X 10 ) derived from (l- C)ascorbic acid in cerebellum. 6

14

0.5n

0

100

200

300

Time (h) Figure 4 continued. Time course of total radioactivity (dmp X 10 ) derived from (l- C)ascorbic acid in eyes. 6

14



Figure 5.

14

6

2

Cumulative C-radioactivity (10 dpm) excreted as C0 . Plotted curve calculated using the empirical model given in Figure 8.


s

>

o

W

o

§

CO

o to

14.

HORNIG

AND

303


H A R T M A N N

T h e p e r c e n t a g e of a d m i n i s t e r e d

1 4

C - r a d i o a c t i v i t y e x c r e t e d as r e s p i r a t o r y

c a r b o n d i o x i d e , w i t h u r i n e a n d feces, a n d t h e t o t a l p e r c e n t a g e

excreted

are g i v e n i n T a b l e I. T h e m e a n values (=b s d ) of the steady state c o n c e n t r a t i o n ( C ) s s

of


a s c o r b i c a c i d i n p l a s m a a n d v a r i o u s organs are s u m m a r i z e d i n T a b l e I I .

Table I. Excreted Feces, of Time

(h)

1 2 6 12 24 48 96 144 192 216 264 312

Percentage of Administered Carbon-14-Radioactivity with Respiratory Carbon Dioxide, with U r i n e and with Respect to Time after O r a l Administration 50 /xCi ( 1 - C ) Ascorbic A c i d to Guinea Pigs 14

CO,

Urine

Feces

Total

9.9 16.5 25.7 30.3 36.9 47.2 58.7 60.8 62.5 63.3

0.2 1.2 1.6 2.6 2.8 4.7 6.5 6.4 6.6 6.8 6.8 6.7

0.3 0.8 1.3 2.0 2.5 2.7 2.8 2.8 2.8 2.6

27.6 33.7 41.0 53.9 67.7 69.9 71.9 72.9

Table II. Mean ± sd of Organ Weight and Steady State Concentration ( C ) of Ascorbic A c i d ( A A ) in Tissues and Mean Content ( X T ) of Ascorbic A c i d in Tissues ss

Organ Weight Organ

Liver Spleen Testes Cerebrum Lungs A d r e n a l glands Parotid gland S u b m a n d i b u l a r glands Kidneys Cerebellum H e a r t muscle Eyes Pituitary gland S u b l i n g u a l glands Plasma

(g) 12.30 ± 0.42 ± 0.84 ± 2.43 ± 2.08 ± 0.11 ± 0.56 ± 0.50 ± 2.57 ± 0.41 ± 0.93 ± 0.77 ± 0.01 ± 0.08 ±

3.2 0.1 0.3 0.2 0.3 0.02 0.1 0.1 0.2 0.05 0.1 0.05 0.002 0.03

Cs S

(mg AA/g of Tissue)

0.33 0.45 0.47 0.28 0.28 1.46 0.48 0.56 0.13

± ± ± ± ± ± ± ± ±

XT

0.07 0.06 0.06 0.06 0.07 0.27 0.02 0.13 0.04

0.0095 ± 0.004°

" E x p r e s s e d i n m g of a s c o r b a t e / m L .


(mg)

4.1 0.2 0.4 0.7 0.6 0.2 0.3 0.3 0.3

304

ASCORBIC

ACID

A l s o g i v e n are the m e a n values ( ± s d ) of the o r g a n w e i g h t s as w e l l as the content [ X T ( m g ) ]

of a s c o r b i c a c i d i n t h e organs, w h i c h h a v e b e e n

e s t i m a t e d b y m u l t i p l y i n g the m e a n steady state c o n c e n t r a t i o n b y

the

mean organ weight.

Evaluation of Kinetic Parameters Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 25, 2018 | https://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch014

Specific A c t i v i t y .

T h e t i m e course of the specific a c t i v i t y of ascor

b i c a c i d i n p l a s m a has b e e n fitted b y a n a n a l o g c o m p u t e r to a s u m of four exponentials: Si =

76.l£T - < + 0

65

1.93e-°- ' + 2 . 7 5 < r 032

00114

' -

80.&T 0

(1)

1

T h e e x p e r i m e n t a l d a t a a n d the c a l c u l a t e d c u r v e are p l o t t e d i n F i g u r e 1. T h e t y p e of E q u a t i o n 1 i m p l i e s a t h r e e - c o m p a r t m e n t system w i t h

first-

o r d e r a b s o r p t i o n , t h a t is, at least three k i n e t i c a l l y d i s t i n g u i s h a b l e pools f o r a s c o r b i c a c i d are reflected i n the p l a s m a . F r o m the d a t a a v a i l a b l e i t 20

0 0

5

10

20

15

30

25

Time (h) Figure 6.

Excretion rate (10 dpm/h) of C-radioactivity 6

14

as CO .


t


14.


cannot

be

decided

305


unambiguously

w h i c h exponential i n E q u a t i o n 1

a c t u a l l y describes the a b s o r p t i o n . I n this case the e x p o n e n t i a l w i t h the largest rate constant is a s s u m e d to be the a b s o r p t i o n t e r m . D e s p i t e the l a r g e s c a t t e r i n g of the ratios of the specific a c t i v i t y i n p l a s m a to the specific a c t i v i t y i n several organs

(values not g i v e n ) , it

appears t h a t e v e n after a t t a i n m e n t of the p s e u d o steady state ( d i s t r i b u t i o n e q u i l i b r i u m ) those ratios are h i g h e r t h a n u n i t y . T h i s suggests

the

specific a c t i v i t y i n p l a s m a to b e h i g h e r t h a n i n tissues. Binding of Ascorbic A c i d in Tissues.

F o l l o w i n g E q u a t i o n 1, the

a p p a r e n t v o l u m e of d i s t r i b u t i o n ( V i ) of the r a p i d l y accessible p a r t of the b o d y ( c e n t r a l c o m p a r t m e n t ) w a s d e r i v e d t o be 0.47 L . F u r t h e r m o r e , a n a p p a r e n t v o l u m e of d i s t r i b u t i o n (V ) S8

(15).

V

M

of 2.2 L has b e e n

estimated

relates the a m o u n t of a s c o r b i c a c i d i n the b o d y i n the steady

state to the c o n c e n t r a t i o n i n p l a s m a . T h e v a l u e of V i a n d V

88

lated assuming 100%

are c a l c u

a b s o r p t i o n of the a d m i n i s t e r e d l a b e l e d


ascorbic

306

ASCORBIC

ACID

a c i d . T h e V i of the c e n t r a l c o m p a r t m e n t m a y b e r e p r e s e n t e d (16,17) E q u a t i o n 2. A n a l o g o u s l y , the V

by

i n t h e steady state w i l l b e d e s c r i b e d b y

88

E q u a t i o n 3.

V

-

x

V

+ ^

P

(2)

V

E


CLE V

SS

= V

1

^V

+

= V

B

P

+ ^V

+

B

OLR

OLE

^V

(3)

B

OLE

T h e m e a n i n g of the s y m b o l s i n E q u a t i o n s 2 a n d 3 are as f o l l o w s : real plasma volume r e a l e x t r a v a s c u l a r v o l u m e i n w h i c h ascorbic a c i d d i s t r i b utes r a p i d l y (even d u r i n g the a b s o r p t i o n phase) v o l u m e of t o t a l b o d y w a t e r V — (V + V) unbound fraction in plasma unbound fraction i n volume V unbound fraction in volume V V + V - f V = v o l u m e of t o t a l b o d y w a t e r

V V

P

E

V a a a V

B

B

P E

P

E

E

R

R

B

P

E

R

T h e u n b o u n d fractions are a v e r a g e d values i n the respective v o l u m e s . B e c a u s e the a p p a r e n t v o l u m e s

of d i s t r i b u t i o n , V i a n d V , ss

l a r g e r t h a n the r e a l p h y s i o l o g i c a l v o l u m e s , the ratios (a /a ) P

E

are

and

much (a /a ) P

R

m u s t b e l a r g e r t h a n u n i t y , t h a t is, a n a p p r e c i a b l y h i g h e r b i n d i n g of ascorbic

a c i d to structures i n the e x t r a v a s c u l a r space c o m p a r e d

with

b i n d i n g to p l a s m a proteins is d e r i v e d . E q u a t i o n s 2 a n d 3 are v a l i d i f there are no active t r a n s p o r t a n d / o r other c o m p l i c a t e d transfer processes. N e g l e c t i n g p a r t i t i o n i n g d u e to p H differences i n v a r i o u s tissues, the c o n c e n t r a t i o n of u n b o u n d a s c o r b i c a c i d is a s s u m e d to be e q u a l i n a l l v o l u m e s i n t o w h i c h the c o m p o u n d is d i s tributed.

A p p l y i n g this p r i n c i p l e to a single tissue, E q u a t i o n 4 c a n b e

derived easily:

OP_ CT_ =

OLT

t^

(4)

p

w i t h the f o l l o w i n g n o t a t i o n s : a a C C

P

T P T

unbound unbound ascorbic ascorbic tissue)

fraction in plasma f r a c t i o n i n tissues acid concentration ( m g / m L plasma) a c i d c o n c e n t r a t i o n ( m g / g tissue « m g / m L

A c c o r d i n g to E q u a t i o n 4 this r a t i o represents a measure for the u n b o u n d f r a c t i o n of ascorbate i n tissues w i t h respect to t h e u n b o u n d f r a c t i o n i n p l a s m a . I n c a l c u l a t i n g the r a t i o of c o n c e n t r a t i o n ( m g / g ) of


14.

HORNIG AND

307


HARTMANN

u n l a b e l e d a s c o r b i c a c i d i n tissues to c o n c e n t r a t i o n ( m g / m L )

i n plasma,

the f r a c t i o n of u n b o u n d a s c o r b i c a c i d i n p l a s m a is a b o u t 1 0 - 6 0

times

l a r g e r t h a n i n tissues. A n e x t r e m e l y h i g h r e l a t i v e b i n d i n g i n t h e a d r e n a l s is r e c o g n i z e d .

L i t t l e is k n o w n a b o u t the b i n d i n g of a s c o r b i c a c i d to

p l a s m a proteins of g u i n e a p i g s

(18).

T u r n o v e r of A s c o r b i c A c i d .

T h e t i m e course of l a b e l e d

ascorbic


a c i d i n organs is r e p r e s e n t e d b y the d i f f e r e n t i a l e q u a t i o n : r\ YT*

Z^-^TuSi-KnXT*

(5)

w i t h the f o l l o w i n g n o t a t i o n s : Si XT* T

specific a c t i v i t y (10 d p m / m g ) of ascorbic a c i d i n p l a s m a a m o u n t (10 d p m ) of C - r a d i o a c t i v i t y i n the o r g a n t u r n o v e r rate ( m g / h ) of ascorbic a c i d f r o m p l a s m a i n t o the organ rate constant ( L / h ) of excretion f r o m the o r g a n 6

6

1T

K

T1

1 4

E q u a t i o n 5 is b a s e d o n the p r e m i s e t h a t e a c h tissue is d i r e c t l y c o u p l e d w i t h the p l a s m a ( m a m m i l l a r y s y s t e m ) .

U s i n g Si f r o m E q u a t i o n

1, t h e d i f f e r e n t i a l E q u a t i o n 5 has b e e n fitted u s i n g a n a n a l o g to the t i m e course of parameters, T

1T

r a d i o a c t i v i t y i n s e v e r a l organs.

and K , T1

are s u m m a r i z e d i n T a b l e I I I . T h e c a l c u l a t e d

curves are s h o w n i n F i g u r e s 2 - 4 . a subjective m e t h o d . ing

of

the

computer

T h e resulting

C u r v e fitting b y a n a n a l o g c o m p u t e r is

T h i s is p a r t i c u l a r l y true i f the r a t h e r l a r g e scatter

e x p e r i m e n t a l d a t a is t a k e n i n t o account.

d e r i v e d values f o r T

Therefore,

the

and K i

s h o u l d b e r e g a r d e d as a p p r o x i m a t i o n s

T h e t u r n o v e r rate (afflux)

of a s c o r b i c a c i d i n t o the o r g a n is d e

1T

T

only. scribed by T . 1T

T h e parameter K

is a f r a c t i o n a l rate constant

T1

c a n b e t r a n s f o r m e d i n t o the t u r n o v e r rate, T

and

(efflux) of a n o r g a n b y

T1

m u l t i p l y i n g i t b y the a s c o r b i c a c i d content ( X T ) of t h a t o r g a n : T

— K

T1

T1

(6)

XT

U n d e r steady state c o n d i t i o n s the efflux T

T1

e q u a l to t h e afflux T . 1T

of a n o r g a n m u s t b e

A s c a n be seen f r o m T a b l e I I I , h o w e v e r , t h e f o r

m a l l y c a l c u l a t e d efflux T n is g e n e r a l l y l a r g e r t h a n t h e afflux T . 1T

d u c i n g the specific a c t i v i t y , S

T

=

Intro

of the tissue i n t o E q u a t i o n 5

XT*/XT,

y i e l d s , after s i m p l e r e a r r a n g e m e n t s : diSr/Si)

—Si

jr

TIT

ST

'-xt~ X KTI


(7)


5

A

0.054

0.25

0.21

1.3

0.02

0.23

0.16

0.01

1.9

0.28

1.0

2.16

0.21

0.75

0.049

0.12

1.92

1.5

0.13

0.14

0.74

4.5

0.31

55.2

( mg/d)

1 T

T /Organ Weight ( mg/d/g)

T

T1

K

2.04

1.80

3.84

0.34

8.88

0.84

1.68

5.04

0.36

0.58

2.16

19.2

(1/d)

Ttot

calculated according to Equation

9;

Tmean

10.

— — — —

calculated according to Equation

1.53

— — — —

1.71

1.29

1.35

2.00

1.77

1.32

1.41

W T .

2.93

0.24

0.27

2.92

0.24

0.23

0.41

78.0

(mg/d)

T u r n o v e r Rates of Ascorbic A c i d and Mean Transit Ti]

TIT calculated according to Equation 5; Tr\ = K \ (Equation 6).

Plasma

Submandibular glands Kidneys Cerebellum H e a r t muscle Eyes Pituitary gland

Liver Spleen Testes Cerebrum Lungs A d r e n a l glands

Organ

TIT

Table III.


14.

HORNIG

AND

309


H A R T M A N N

I n t h e p s e u d o steady state ( d i s t r i b u t i o n e q u i l i b r i u m ) , the r a t i o of t h e specific activities ( S i / S i ) is i n d e p e n d e n t w i t h t i m e , w h i c h leads t o T

E q u a t i o n 8.


S\ ST

Tri

^gj

TIT

T h i s result suggests t h a t the o b s e r v e d r a t i o of specific activities a n d t h e r a t i o of t u r n o v e r rates

(Table

III)

parallel each

other

according

to

E q u a t i o n 8. T h e t o t a l t u r n o v e r of a s c o r b i c a c i d (T )

i n t h e b o d y equals

tot

the

i n t a k e of a s c o r b i c a c i d ( m g / d ) u n d e r steady state c o n d i t i o n s a n d a s s u m ing 100% bioavailability. T

tot

c a n also b e c a l c u l a t e d b y t h e o c c u p a n c y

p r i n i c i p l e ( 1 9 ) u s i n g the t i m e course of r a d i o a c t i v i t y i n o r g a n s : „ dose • XT dose 1u GO f* CO

/ XT* w i t h the f o l l o w i n g

dt

J

S

(9)

dt

T

notations:

t o t a l t u r n o v e r rate (mg of a s c o r b a t e / d ) i n the b o d y a d m i n i s t e r e d C - r a d i o a c t i v i t y (10 d p m ) a m o u n t (mg) of ascorbate i n t h e respective organ i n steady state a m o u n t (10 d p m ) of C - r a d i o a c t i v i t y i n the o r g a n specific a c t i v i t y (10 d p m / m g of ascorbate) i n tissues

Ttot

dose XT

1 4

XT* S

6

6

1 4

6

T

C o n s e q u e n t l y the t o t a l t u r n o v e r i n p l a s m a c a n b e o b t a i n e d b y i n t r o d u c i n g the p l a s m a specific a c t i v i t y (Si, E q u a t i o n 1) The T

t o t

i n t o E q u a t i o n 9.

values c a l c u l a t e d u s i n g E q u a t i o n 9 are l i s t e d i n T a b l e I I I . T h e s e

values m a y be c o m p a r e d w i t h the d a i l y i n t a k e of a s c o r b i c a c i d present i n the diet, w h i c h w a s d e t e r m i n e d to be 16 ±

3 mg.

T h e specific a c t i v i t y i n p l a s m a was f o u n d to b e h i g h e r t h a n t h a t i n tissues, therefore t h e t o t a l t u r n o v e r d e r i v e d f r o m p l a s m a is l o w e r t h a n t h e t u r n o v e r d e r i v e d f r o m r a d i o a c t i v i t y i n tissues. R e l e v a n t k i n e t i c parameters

(half-life, b o d y pool, a n d mean transit

t i m e i n o r g a n s ) c a n b e c a l c u l a t e d . A c c o r d i n g to E q u a t i o n 1 t h e specific a c t i v i t y i n p l a s m a shows a t r i p h a s i c d e c a y w i t h h a l f - l i v e s of t± = t

2

=

22 h , a n d t = s

1.1 h ,

61 h . T h e h a l f - l i v e s * i a n d t essentially d e s c r i b e t h e 2

d i s t r i b u t i o n of t h e c o m p o u n d i n t o the system. T h e t h i r d h a l f - l i f e of 61 h (2.5 d ) is v a l i d for a l l tissues after a t t a i n m e n t of t h e d i s t r i b u t i o n e q u i l i b r i u m a n d represents t h e o v e r a l l h a l f - l i f e of e l i m i n a t i o n f r o m t h e b o d y u n d e r the s p e c i a l c o n d i t i o n s of the s t u d y (ascorbate status of the a n i m a l s ) .


310

ASCORBIC

T h e a m o u n t of a s c o r b i c a c i d i n the b o d y ( b o d y p o o l ) c a n b e t a i n e d b y m u l t i p l y i n g t h e v o l u m e of d i s t r i b u t i o n (V ) ss

steady state c o n c e n t r a t i o n [C

(Table II)]

ss

ACID

ob

b y the a v e r a g e d

of a s c o r b i c a c i d i n p l a s m a .

T h i s c a l c u l a t i o n y i e l d s a b o d y p o o l of 21 m g .

U n f o r t u n a t e l y , t h e esti

m a t e d b o d y p o o l c a n n o t b e c o m p a r e d w i t h a n e x p e r i m e n t a l v a l u e since the t o t a l p o o l w a s not accessible e x p e r i m e n t a l l y .


T h e m e a n transit t i m e

(t

m e a n

of

)

1 4

C - r a d i o a c t i v i t y i n organs

f o r m a l l y b e e n e v a l u a t e d u s i n g E q u a t i o n 10 w i t h X T * as t h e

1 4

has

C-radio-

activity i n the organs: CO

t • XT*

/ ^mean

dt

~

= =

J

(10) XT*

dt

T h e r e s u l t i n g p a r a m e t e r s are g i v e n i n T a b l e I I I . T h e r e s u l t i n g m e a n t r a n s i t t i m e m a y b e u s e d as a measure of the residence t i m e of a m o l e c u l e i n a n o r g a n c o m p a r e d w i t h a s e c o n d o r g a n , regardless of p o s s i b l e r e c i r c u l a t i o n . R a n k i n g the organs c o r r e s p o n d i n g to these m e a n t r a n s i t times ( T a b l e I I I ) three groups m a y b e d i s t i n g u i s h e d : g r o u p 1 i n c l u d e s l i v e r , k i d n e y , l u n g s , a n d h e a r t m u s c l e w i t h m e a n t r a n s i t times of 7 1 - 7 5 ; g r o u p 2 i n c l u d e s spleen, p i t u i t a r y g l a n d , eyes, a n d a d r e n a l g l a n d s w i t h m e a n t r a n s i t times of 8 1 - 8 4 h ; a n d g r o u p 3 i n c l u d e s s u b m a n d i b u l a r g l a n d , testes, c e r e b r u m , a n d c e r e b e l l u m w i t h m e a n t r a n s i t times of 9 9 - 1 4 2 h . classification agrees f a i r l y studies (10

the

results of

The

autoradiographic

(7,9,10).

Excretion of 6

well with

dpm/h)

of

1 4

14

C-Radioactivity.

T h e m a x i m u m rate of

excretion

C - r a d i o a c t i v i t y as c a r b o n d i o x i d e is r e a c h e d at a b o u t

0.5 h ( F i g u r e 6 ) . H o w e v e r , the p e a k i n a c t i v i t y vs. t i m e curves of p l a s m a a n d of the r a p i d l y p e r f u s e d organs l i k e l i v e r , l u n g s , k i d n e y is at a b o u t 1.5 h ( F i g u r e 2 ) . T h e excretion of

14

C - r a d i o a c t i v i t y as l a b e l e d c a r b o n d i o x i d e

is not d i r e c t l y r e l a t e d to p l a s m a i n the first t i m e p e r i o d after a d m i n i s t r a t i o n of the l a b e l . A n i n t e r m e d i a t e c o m p a r t m e n t X 3 m u s t b e assumed. I n the s e c o n d t i m e p e r i o d after a d m i n i s t r a t i o n i t w a s not p o s s i b l e to d e s c r i b e the d e p e n d e n c y of excreted r a d i o a c t i v i t y o n t i m e b y r e l a t i n g i t to the t i m e course of specific a c t i v i t y i n p l a s m a or b y r e l a t i n g i t to the t i m e course of r a d i o a c t i v i t y i n organs. T h e r e f o r e , a s e c o n d i n t e r m e d i a t e c o m p a r t m e n t X 2 m u s t be i n t r o d u c e d . T h e c o m p l i c a t e d e x c r e t i o n p a t t e r n w a s finally r e p r e s e n t e d b y a n e m p i r i c a l m o d e l ( F i g u r e 8 ) .



6

14

s

14

K2A=0.95

K1A=0.88

14

K3A=6.0

Figure 8. Empirical model for excretion of C-radioactivity in guinea pigs. Key: D l , D 2 , D 3 , fractions of administered C-radioactivity in the intermediate compartments 2 and 3; C , U , F , C-radioactivity (10 dpm) excreted as CO with urine and feces; T , turnover rates (mg/)h; K , rate constants (L/h).

D2=48

M

D3=24


312

ASCORBIC

ACID

T h e a d m i n i s t e r e d dose is s u b d i v i d e d i n t o three p a r t s . D l denotes the f r a c t i o n of t h e e x c r e t i o n t h a t is l i n k e d to t h e t i m e course of specific a c t i v i t y ( S i ) i n p l a s m a . T h e a m o u n t s D 2 a n d D 3 a r e fractions a p p e a r i n g i n the intermediate compartments X 2 a n d X 3 , respectively. T h e parameters fitting

of this e m p i r i c a l m o d e l h a v e b e e n e v a l u a t e d b y

the experimental data b y using a n analog computer.

T h e model


has b e e n v e r i f i e d a n d t h e p a r a m e t e r estimates refined b y s i m u l a t i o n s (program C S M P ) on a digital computer.

T h e r e s u l t i n g parameters a r e

s u m m a r i z e d i n F i g u r e 8. T h e c a l c u l a t e d curves a r e c o m p a r e d w i t h t h e e x p e r i m e n t a l d a t a i n F i g u r e s 5 a n d 6. B e c a u s e of t h e c o m p l e x i t y of t h e system, t h e p a r a m e t e r

set, a n d even t h e m o d e l

itself, m i g h t n o t b e

u n i q u e ; another m o d e l d e s c r i b i n g t h e d a t a e q u a l l y w e l l m i g h t exist. T h e excretion of

14

C - r a d i o a c t i v i t y w i t h u r i n e is also represented b y

the e m p i r i c a l m o d e l ( F i g u r e 8 ) . T h e c a l c u l a t e d c u r v e of t h e c u m u l a t i v e 14

C - r a d i o a c t i v i t y i n u r i n e is s h o w n i n F i g u r e 7 together w i t h t h e e x p e r i

mental data. A c c o r d i n g to the empirical model ( F i g u r e 8) the calculated curve of

the cumulative

13

C - r a d i o a c t i v i t y i n feces is p l o t t e d

t h e d a t a points i n F i g u r e 7 ( l o w e r c u r v e ) .

together

with

A fraction, w h i c h might be

d u e t o t h e l a r g e s a m p l i n g i n t e r v a l s i n t h e first p e r i o d after i n t a k e of t h e l a b e l , w a s excreted f r o m t h e i n t e r m e d i a t e c o m p a r t m e n t X 3 b u t c o u l d n o t be

detected.

Discussion T h e fate of a s c o r b i c a c i d a d m i n i s t e r e d o r a l l y as ( 1 - C ) a s c o r b i c a c i d 1 4

has b e e n f o l l o w e d i n t h e g u i n e a p i g . T h e d a t a o n t h e o v e r a l l e x c r e t i o n of l a b e l e d m a t e r i a l ( T a b l e I ) c o n f i r m e a r l i e r reports d e m o n s t r a t i n g t h e r e s p i r a t o r y p a t h w a y to b e t h e m a j o r route of c a t a b o l i s m i n the g u i n e a p i g (3,20,21).

A s c o r b i c a c i d is r a p i d l y a n d w i d e l y d i s t r i b u t e d t h r o u g h o u t

the b o d y (4).

W e h a v e a t t e m p t e d to d e t e r m i n e t h e r e m a i n i n g p a r t o f

the l a b e l i n t h e tissues to o b t a i n a b a l a n c e u s i n g t h e t o t a l excreted r a d i o a c t i v i t y . I t w a s n o t p o s s i b l e e x p e r i m e n t a l l y to d e t e r m i n e t h e r e m a i n i n g l a b e l i n s t o m a c h , intestine, a n d b o n e .

I n a d d i t i o n , o n l y estimations o f

r a d i o a c t i v i t y i n f a t , skeletal m u s c l e , a n d s k i n w e r e p o s s i b l e , b e c a u s e t h e t o t a l mass w a s n o t a v a i l a b l e . T h e r e f o r e , t h e t o t a l b o d y p o o l of a s c o r b i c a c i d i n our investigation cannot

b e assessed

a n d compared

with the

c a l c u l a t e d v a l u e of 21 m g . H o w e v e r , figures f r o m t h e l i t e r a t u r e suggest that, w i t h d a i l y intakes of 500 m g of a s c o r b i c a c i d / k g of diet, a b o d y p o o l of this size is a c h i e v e d

(22-24).

T h e r a t i o of t h e specific a c t i v i t i e s i n p l a s m a a n d tissues w e r e l a r g e r t h a n u n i t y e v e n after a t t a i n m e n t of the steady state; this finding c o u l d b e c a u s e d b y tissue ascorbate n o t e x c h a n g i n g w i t h t h e i n t r o d u c e d , l a b e l e d


14.

HORNIG

AND

a s c o r b i c a c i d w i t h i n the t i m e p e r i o d of the e x p e r i m e n t . exchange

suggests

313


H A R T M A N N

the existence

of

T h e failure to

structures i n tissues f r o m

which

a s c o r b i c a c i d is s l o w l y released. I f this s u p p o s i t i o n is v a l i d the c a l c u l a t e d v a l u e of the t o t a l b o d y p o o l w o u l d be u n d e r e s t i m a t e d . T h e f r a c t i o n of u n b o u n d a s c o r b i c a c i d i n p l a s m a w a s f o u n d i n o u r s t u d y t o b e u p to sixty times l a r g e r t h a n t h a t i n tissues. T h e a d r e n a l s


e x h i b i t a n e x t r a o r d i n a r i l y h i g h r e l a t i v e b i n d i n g (170 times l a r g e r t h a n p l a s m a ) , w h i c h confirms t h e o b s e r v a t i o n t h a t several p r o t e i n fractions c a n b e i s o l a t e d f r o m adrenals b y S e p h a d e x c h r o m a t o g r a p h y ; a c i d w a s f o u n d to b e b o u n d to these fractions ( 2 5 ) .

ascorbic

T h e lower relative

b i n d i n g i n c e r e b r u m a n d l u n g s supports nonspecific b i n d i n g of

ascorbic

a c i d to g u i n e a p i g b r a i n h o m o g e n a t e s ( 2 6 ) a n d to rat l u n g h o m o g e n a t e s (27).

T h e r e l a t i v e b i n d i n g of a s c o r b i c a c i d w a s also l o w i n l i v e r , i n

accordance

w i t h data reported earlier (28).

H o w e v e r , a rather w i d e

c o n t r o v e r s y exists o n the b i n d i n g of a s c o r b i c a c i d to p r o t e i n .

Several

authors h a v e suggested that a s c o r b i c a c i d is b o u n d to p r o t e i n i n a d r e n a l s a n d i n l i v e r tissue (25, 29) such b i n d i n g (28).

w h e r e a s others f o u n d n o e v i d e n c e

for any

A l s o , no evidence for protein-bound ascorbic

c o u l d b e f o u n d i n other tissues ( b r a i n , l u n g )

acid

(26,27).

D i f f e r e n t concentrations i n c o m p a r t m e n t s t h a t c a n exchange

ascorbic

a c i d m a y also b e a c h i e v e d b y n o n l i n e a r transfers s u c h as a c t i v e t r a n s p o r t processes. N o n l i n e a r i t y , h o w e v e r , c a n n o t b e d e t e r m i n e d b y e x p e r i m e n t a l designs u s i n g o n l y one steady state l e v e l . T h e b r a i n , a d r e n a l s , p i t u i t a r y g l a n d , a n d eyes take u p a s c o r b i c a c i d b y a n e n e r g y - d e p e n d e n t t r a n s p o r t m e c h a n i s m (30,

active

31).

O u r e x p e r i m e n t a l d e s i g n a l l o w e d the e s t i m a t i o n of a s c o r b i c t u r n o v e r i n v a r i o u s tissues ( F i g u r e s 2 - 4 ) . per unit time (turnover) (T

i r

= 4.5 m g / d / g

mg/d)

acid

T h e a m o u n t of a s c o r b i c a c i d

r e a c h i n g the tissues was h i g h e s t i n the l i v e r

of tissue, c o r r e s p o n d i n g to a t o t a l t u r n o v e r of

f o l l o w e d b y a d r e n a l s , testes, a n d p i t u i t a r y g l a n d ( T a b l e

55

III).

T h e r a t h e r h i g h t u r n o v e r i n t h e l i v e r e x c e e d i n g the t o t a l t u r n o v e r m a y b e c a u s e d b y a m u l t i p l e r e c i r c u l a t i o n of a s c o r b i c a c i d to this o r g a n . T h e t u r n o v e r rates of a s c o r b i c a c i d f r o m tissues (afflux T i) T

l a r g e r t h a n the afflux (see T a b l e I I I , c o l u m n h e a d e d

Kinetic Behavior of Ascorbic Acid in Guinea Pigs - American Chemical

Recommend Documents