In vivo sulfation of cholesterol by ascorbic acid 2-sulfate

Atherosclerosis, 17 (1973) 37-48 0 Elsevier Scientific Publishing Company, Amsterdam

IN VIP’0 SULFATION

OF CHOLESTEROL

A. J. VERLANGIERI

R. 0. MUMMA**

AND

37 - Printed in The Netherlands

BY ASCORBIC ACID 2-SULFATE*

Pesticide Research Laboratory and Graduate Study Center, Departments of Biochemistry and Entomology, The Pennsylvania State University, University Park, Pa. 16802 (U.S.A.) (Revised, received February 22nd, 1972)

SUMMARY

Rats were injected via cardiac puncture with an equivalent radiolabeled amount of s5S04’ (Group I), L-ascorbic acid (20 mg) plus 35S04’ (Group II), and s5S-Lascorbic acid 2-sulfate (20 mg) (Group III) in order to investigate the relationship between these compounds and s%-cholesterol sulfate excretion. Urine and feces were analyzed periodically. Blood and adrenal glands were analyzed at the termination of the experiment (45-48 h). Total W-activity, %-cholesterol sulfate, 3sS-inorganic sulfate and %-organic sulfate were quantified. The %-cholesterol sulfate was found primarily in the feces in the following yield based upon the injected %-dose: I, 0.15 % ; II, 0.3 % ; III, 7.1%. Both L-ascorbic acid and L-ascorbic acid 2-sulfate elevated the radiochemically labeled cholesterol sulfate excreted (2 x and 50 x, respectively). These data prove the existence of a %-sulfate transfer from %-ascorbic acid 2-sulfate to cholesterol resulting in the formation of a5S-cholesterol sulfate, whether direct or indirect, and may in part explain the reported, but controversial, hypocholesterolemic effects of L-ascorbic acid.

Key words:

Ascorbic acid - Ascorbic acid 2-sulfate - Cholesterol - Cholesterol sulfate - Cholesterol sulfate excretion - Fecal cholesterol sulfate extraction - Hypocholesterolemic - In vivo sulfation - Sulfation mechanism

* This work was supported in part by U.S. Public Health Service (Grant AM08481) and by The Pennsylvania Agricultural Experiment Station. Given in part at the 55th Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, ill., April 12-16, 1971l. ** To whom correspondence should be made.

A. J. VERLANGIERI,

38

R. 0.

MUMMA

INTRODUCTION

A number of investigators (AA) and cholesterol metabolism. much controversy. Administration serum cholesterol

have inferred a relationship between ascorbic acid The true significance is yet unresolved and open to of AA to animals has resulted in the lowering of

levels, the removal

and other beneficial

relationships

of cholesterol

deposits

2- 12. A hypercholesterolemic

from the arterial

intima,

relationship

with AA

has also been reportedi2J3. In the two cases cited the authors have suggested the higher serum cholesterol levels are owing to mobilization of cholesterol. Other investigators have reported

no significant

relationship

between

AA and cholesterol

in animals14-1s.

For many years the existence of cholesterol sulfate (CS) in animal tissues and fluids has been known. Its presence has been established in human and bovine adrenal glands and in adrenal tumorsl7, in human plasma, bile, urine, kidney and larger quantities in feceslsllg. Several young human disorders, were found to excrete approximately (wet wt.)lg. CS comprises 80-85 % of the sulfate reported to vary for adults between 14-85 mg

subjects suffering from neurological 100 mg of crude CS in 80 g of feces fraction in human feces and has been per dayaeJ1. It was thought that CS

was an end product of metabolism, however, it is evident now that the CS pool is at least partially involved in steroid biosynthesisl7. Cholesterol has been sulfated by a partially purified enzyme preparation obtained from guinea pig liver and 3’-phosphoadenosine S-phosphosulfate (PAPS)22*2s. It is not known at the present time if this is the only mechanism of biological sulfation. In light of these data we have turned our interest to L-ascorbic acid 2-sulfate (AAS). Mumma synthesized AAS and demonstrated its excellent sulfating potential under mild oxidizing conditions and under transesterification conditions. Octyl sulfate and CS were synthesized in this manner. We and others have proposed that AAS may be involved in biological sulfationas~ss and in support of this hypothesis we have recently isolated AAS from selected rat organsaT. The observed, but controversial, hypocholesterolemic effect of AA, may be in part its influence on the biosynthesis of CS. Accordingly, we undertook a radiochemical investigation to determine if AAS could act as a sulfating agent in vivo and transfer its 35S04= to cholesterol resulting in the formation of ssS-CS. To accomplish this, rats were injected via cardiac puncture with an equivalent radiolabeled amount of 3504’ (Group I), AA (20 mg) plus 35SO4’ (Group II and IIA) and s%-AAS (20 mg) (Group III and IIIA). The feces, urine, blood and adrenal s%-CS, %-inorganic sulfate and glands were analyzed for total ssS-activity, s%-organic sulfate. Group III and IIIA rats possessed greatly elevated s5S-CS levels in the feces and these data proves the existence of a 3YS-sulfate transfer from s%-AAS to cholesterol resulting in the formation of 35S-CS. EXPERIMENTAL

Materials All solvents

were redistilled.

AA was purchased

from Eastman

Organic

Che-

h

ViVO

SULFATION

OF CHOLESTEROL

BY ASCORBIC

ACID Z-SULFATE

39

micals and ssSO4’ from New England Nuclear Corporation. CS was synthesizedQspQQ and used as a standard in the various chromatographic techniques. Q%-AAS was also synthesized according to Mumma et al.3O. Chromatography

Thin-layer (TLC), paper (PC) and column chromatography were used for qualitative and quantitative analyses. Two TLC adsorbents were used: Supelcosil 12B (Supelco, Inc.) and Eastman Chromagram Sheet (Distillation Product Industries). Several solvent systems were used in development of the thin-layer plates: chloroformmethanol-water (65 :25 :4, v/v/v), diethyl ether-benzene-ethanol-acetic acid (40 :50 :2 : 0.2, by vol.)31 and chloroform-methanol-water-acetic acid (65 :50 : 15 : 1, by vol.). Paper chromatography utilized Whatman No. 4 paper and the following solvent systems: phenol-water (100:40, w/w) and n-butanol-propionic acid-water (10 :7 :5, v/v/v). Sephadex LH-20 (Pharmacia Co.) was used in the column chromatographic procedure for the isolation of CS utilizing a chloroform-methanol (1 :l, v/v) solvent system32. CS was visualized on TLC with a 20 % aqueousp-toluenesulfonic acid spray and by two non specific techniques: (a) ultraviolet detection employing an adsorbent containing a zinc silicate phosphor and (b) a sulfuric acid char. The 35S-labeled compounds were located by radioautography using Kodak Royal Blue Sensitive X-ray film. A 1% methanolic ferric chloride spray30 was used for visualization of AAS on TLC and PC. Experimental design

Male rats (Wistar) weighing approximately 150 g were placed in metabolism cages and injected, via cardiac puncture (5 cc Luer-Lok disposable syringe, 1.5 inch 22 gauge needle) with the experimental solutions, under light ethyl ether anethesia. Rats were provided tap water and fed a standard pelleted diet (Purina Co.) ad l&turn. A 3 day acclimatization time was used before injection. Feces and urine were collected separately during the course of the experiment (45-48 h). In a preliminary experiment investigating the toxicity of AAS, 6 rats were injected intracardially with 0.0, 0.25, 0.50, 7.0, 15.0 and 25.0 mg of the potassium salt of AAS dissolved in 0.5 ml of saline, respectively. After 7 days the rats were sacrificed and the lungs, heart, kidneys, liver and stomach were examined and no gross pathology was observed. Group I rats (3) were injected with approximately 45 ,&i of carrier free 3sS04= dissolved in 1.5 ml saline (15 ,uCi of 35S04= per 0.5 ml saline per rat). This same procedure was used for Group II rats (3), except that 60 mg of AA was added to the 1.5 ml saline before injection (15 ,uCi of 35S04= plus 20 mg AA per 0.5 ml saline per rat). Group IIA rats (5) were injected with 100 mg of AA plus 1.65 mCi of 35S04= prepared as above (330 ,uCi of 35S04= plus 20 mg AA per 0.5 ml saline per rat). Group III rats (3) were injected with 60 mg (45 @i) of the sodium salt of Q%-AAS (15 &i of Q%-AAS per 0.5 ml saline per rat). Group IIIA rats (5) were

40

A. J. VERLANGIERI,

injected with 100 mg (5 ,uCi) a%-AAS pared in the same manner.

(1 $i

R. 0.

MUMMA

s5S-AAS per 0.5 ml saline per rat) pre-

Sample collection After injection, feces and urine were collected separately at the same time of day at 4,20,30 and 45 h (Groups I, II, and III) and at 24 and 48 h (Groups IIA and IIIA). Before each subsequent collection the rat-cage troughs were rinsed with distilled

water and the rinse water was collected.

The feces and urine samples

were

then pooled according to group, placed in plastic screw top bottles and stored at -40°C. At the termination of the experiment (4548 h) the rats were anesthetized with ethyl ether. The blood was collected via cardiac exsanguination (10 cc Luer-Lok disposable syringe, 1.5 inch 22 gauge needle), pooled according to group, allowed to clot and serum collected. Adrenal glands were then immediately excised via abdominal incision. The individual rat carcasses were placed in plastic bags and stored at -40°C. Urine analysis The total 35S-activity in the urine at each time interval in Group I, II and III was analyzed. All sample volumes from each group at each time interval were measured. Triplicate 50 ~1 aliquots were then placed in scintillation vials containing 15 ml Aquasol and counted for 5 min. In addition an aliquot of each urine sample was extracted with n-butanol and the %-activity of the aqueous, and n-butanol fractions was determined. Urine was also analyzed for percentage composition of s%-inorganic sulfate and 3sS-organic sulfate by a barium chloride precipitation method. Urine samples and two aliquots of ssSO4’ (as controls) were placed in 15 ml centrifuge tubes. Sodium sulfate (0.5 g) was added to each tube, followed by sufficient amount of barium chloride to effect complete precipitation of sulfate ion. The tubes were mixed thoroughly with a Vortex and then centrifuged at 600 r.p.m. for 0.5 h. Barium sulfate (10 mg) was then added to each tube and the tubes again centrifuged for 0.5 h. The supernatant was filtered, placed in another 15 ml centrifuge for an additional 18 h. After recentrifugation 50 ,ul aliquots and counted for s?%organic sulfate.

tube and allowed to stand were removed in triplicate

Feces analysis Feces were pooled from 0 to 20 h and, from 20 to 45 h in Groups I, II and III and from 0 to 24 h and from 24 to 48 h in Groups IIA and IIIA and then analyzed for total s%-composition. A weighed amount of feces approximately one half of the total, was homogenized in a Virtis Macro Homogenizer for 0.5 h in chloroform-methanol (2:1, v/v). The homogenate was filtered through a sintered glass funnel containing a previously tared glass fiber filter mat. The insoluble residue was dried, weighed and an aliquot (3&60 mg) was counted in a liquid scintillation counter. The filtrate was

h

ViVO SULFATION

evaporated

OF CHOLESTEROL

BY ASCORBIC

to dryness with a rotary-flash

in 140 ml chloroform-methanol

41

ACID Z-SULFATE

evaporator

and the residue was resuspended

(2:1, v/v) and washed according

to Folch et ~1.33.

The lower chloroform phase was evaporated to dryness and the residue resuspended in 2 ml of chloroform-methanol (1: I, v/v). This solution was then placed on a 25 g LH-20 Sephadex column (22 mm x 22 cm) prepared in chloroform-methanol (I :l, v/v). The flow was adjusted to 0.1 ml/min and thirty 5 ml fractions were collected.

Each fraction

was evaporated

to dryness

under

nitrogen.

The residue

was

redissolved in 0.5 ml chloroform-methanol (1 :l, v/v). An aliquot of each fraction (20 ,ul) was analyzed by TLC. Synthetic ssS-CS was used as a standard. The TLC plates were developed in chloroform-methanol-water (65 :25 :4, v/v/v). The position of the compounds were visualized by UV and by a p-toluenesulfonic acid char. The position of the s%-labeled compounds on these thin-layer plates was also determined by radioautography. Fractions 13-17 contained the 35S-CS and represented >80% of the total s5S-activity placed on the column. They were pooled and evaporated to dryness. The residue was redissolved in chloroform-methanol (1 :l, v/v) and 50 ~1 aliquots in triplicate were counted. Periodically the elution pattern of the LH-20 Sephadex column was checked with standard s%-CS and routinely > 98% recovery of 35S-CS was obtained. Adrenal gland analysis The procedure for the isolation of steroid conjugates from the adrenal gland was essentially that of DeMeio et al. 34. The glands (two from each Group) were homogenized in 0.9% NaCl (2 ml). The homogenate was deproteinized with 6 ml of the cold 80% evaporated in 6 ml of ethyl ether

ethanol. After to dryness with distilled water. and then with

35SO4’ was removed previously described. n-butanol and ethyl TLC

separation of the precipitate the ethanolic extract was a rotary-flash evaporator and the residue was redissolved This aqueous solution was extracted first with 0.5 ml of 1.0 ml of n-butanol saturated with water. The inorganic

from the aqueous phase by a barium chloride precipitation as Aliquots (50 ~1) were removed in triplicate from the aqueous, ether fractions and counted. Aliquots were also analyzed by

(chloroform-methanol-water-acetic

(phenol-water;

100:40,

w/w and

acid;

65 :50: 15 : 1, by vol.),

n-butanol-propionic

acid-water;

and

by PC

10:5:7,

v/v/v).

Serum analysis Serum was analyzed for total s%-activity and for 35S-CS. The 3%S-CS was isolated and quantified in the same manner as that used for the feces. Scintillation counting A Tri-Carb Model 314AX Liquid Scintillation Spectrometer (Packard Instrument Co.) was used to count the samples. Efficiencies were calculated by the channel ratio method. All samples were counted in aliquots of at least 50 ,ul in triplicate. Premixed liquid scintillator, Aquasol, was purchased from New England Nuclear Corp. and used in all analyses and afforded little quenching with aqueous samples.

42

A. J. VERLANGIERI, R. 0. MUMMA

FECES

HOMOGENIZE CM 2:1

-

evaporate CM I:1

LH-20 * SEPHADEX COLUMN

* IDENTIFY

filter

p

““S -CS

FOLCH

WASH

5ml FRACTIONS

L -

combine

FRACTIONS

T.L.C.forCS

SCINTILLATION COUNTING

Fig. 1. Scheme of isolation of %&cholesterol

sulfate from feces.

RESULTS AND DISCUSSION

The blood, adrenal glands, feces and urine were examined for their 35S-CS composition. The feces contained 99% of ssS-CS quantified. A small amount was found in the blood (cu. 0.5 %) and traces in the urine. It was not detected in the adrenal glands. The experimental scheme for the isolation of ssS-CS from the feces is shown in Fig. 1. The weight of the feces and the percentage ssS-CS excreted in the feces relative to the injected %-dose is presented in Table 1. Group I rats (3%04=) excreted a small percentage of the injected s%-dose as s5S-CS (0.11 ‘A in 20 h). Although the relative excretion of 3sS-CS by Group II rats (AA plus ssSO4’) was doubled compared to Group I the statistical significance is unknown. Group III rats (s%-AAS) greatly increased the excretion of s5S-CS relative to the injected 3sS-dose, (5.20-6.9 %) in the first 20 h, approximately 50 times the background (Group I). The same trend was also seen in the next 25 h although not as dramatically. The high initial rate of excretion of ssS-CS in Group III when compared to the other two groups suggests a different mechanism of biosynthesis of ssS-CS in Group III. In any case, sulfation of cholesterol by s5S-AAS seems to be independent of or ancillary to the PAPS mechanism utilizing inorganic a%-sulfate. At the end of the experiment ssS-CS was found TABLE

1

PERCENTAGE

%-CS

Group

I (3%04=)

II (AA plus 3%04-) III (a%-AAS)

IIA (AA plus

EXCRETED IN THE FECES RELATIVE TO INJECTED 35S-~~~~

No. rats

3 3 3

35S04=)5 IIIA (a%-AAS) 5

B Wet weight.

Feces wt.

(g) a

y* 35s-cs

O-20 h

20-45 h

O-20 h

20-45 h

14.66 20.17 18.14

18.39 28.15 23.32

0.11 0.21 5.20

0.05 0.04 0.95

O-24 h

24-48 h

O-24 h

24-48 h

22.76 18.48

17.38 18.02

0.22 6.90

0.11 1.20

Total % =s-cs

0.16 0.25 6.15

0.33 8.10

h

ViVOSULFATION OF CHOLESTEROL BY ASCORBIC ACID Z-SULFATE

5

IO

1

I

I

I

I

I

I

I5

20

25

30

35

40

45

43

HOURS Fig. 2. Urinary excretion profiles of total 35S-activity.

in the blood of all the rats invery low levels. Serum from Group III rats contained 0.05 % 35S-CS relative to the injected s5S-dose, about eight times that found in Group I rats. Since the feces of Group III rats contained elevated 35S-CS levels, it is consistent that the blood of Group III rats would also have elevated ssS-CS levels. Blood CS has been reported

to be excreted primarily

in the feces3s.

Total 35S-activity

Fig. 2 summarizes the total 35S-activity in the urine of Groups I, II and III. There was no significant urine volume difference between the groups. The accumulated urine from Group I rats contained 99.5 % of the injected s%-dose. Somewhat less, 96.5 % was excreted in Group II rat urine. Group III rats excreted only 60.2 % of the injected %-dose in 45 h. When the 3sS composition of the urine, 60.2’?, and feces, 6.15 % are combined 66.35 % of the injected 3% dose can be accounted for in Group III animals. Approximately 34% of the s5S-dose remained in the rat carcasses and may represent in part additional sulfation. Groups I and II rats had very similar excretion profiles of 35S-activity in their urine (Fig. 2). The excretion pattern of Group III rats was significantly different; since its rate of excretion initially was greater than the other two. However, in contrast, after 20 h very little additional excretion of %-activity was observed. If ssS-AAS was initially being hydrolyzed or oxidized to ascorbic acid and ssSO4’, one would expect the excretion profile to be the same as Group II. This was not observed and suggests that undegraded s?S-AAS must be responsible for the enhanced incorporation of %-activity into the ssS-CS. The difference in the excretion profiles suggests that the ssS-labeled material excreted in Group III rats may not be the same as that excreted by the other groups. This will be clarified in the next section. The excretion rates of Group I and II rats were rather constant while that of Group III fell off sharply with time (Fig. 2). The amount of carrier free 35S04’, injected into rats of Group I and II is small compared to the total body SOS= pool and

A. J. VERLANGIERI, R. 0. MUMMA

44

would exchange freely with the pools, resulting in constant release in the urine. The high initial rate of s%-excretion in Group III may be explained by: (1) AAS titer exceeded the threshold level of the kidney, (2) that s5S-AAS is not as permeable to cell membranes as 3sS04= or, (3) s%-AAS might remain in the extracellular fluid where exchange from the blood to kidney is most rapid. The decrease in the excretion rate of 35S-AAS (after 20 h) might be explained by: (1) AAS being chemically decomposed, (2) AAS may only be absorbed when extracellular titer reaches a certain level, or (3) AAS may have a target tissue where it accumulates with time. The other fractions of the feces were also analyzed for general 35S-activity; namely, the Folch wash upper phase (water solublesss), and the residue (non lipid extractables). The water soluble fraction contained the following s%-activity based on the injected ssS-dose, Group I-l .4 %, Group II-O.6 %, and Group III&O.1%. The residue material accounted for approximately 0.4% in all groups. 35S-Organic versus 35S-inorganic sulfate

The 35S-activity of the urine in Groups I, II and III was separated into 3% organic sulfate and %-inorganic sulfate by a barium chloride precipitation and these data are presented in Table 2. Control samples possessing only s5S-inorganic sulfate were essentially 100 % precipitated with barium chloride. The urine of Group I and II contained 84.2 and 83.7% of the s5S-activity in the urine in the form of 35S04=, respectively. Urine from Group III rats gave a completely different picture; 87.3% of the %-activity was in the form of %-organic sulfate. It is interesting to note that 79.5 % of the total %-organic sulfate in the urine of Group III was excreted in the first 4 h after injection. Further investigation proved most of this %-activity to be intact ssS-AAS which had been excreted unaltered into the urine, apparently surviving the physiological conditions of the rat. The s%-organic sulfate fraction of the urine was further characterized by an extraction with n-butanol. The n-butanol extract, which contains the steroid sulfates, possessed 4.5 % of the ssSactivity of the organic sulfate fraction of Group III rats.

TABLE

2

PERCENTAGE

OF

35s-ORGANIC

AND

35s-INORGANIC

SULFATE

COMPOSITION

OF

TOTAL

35S-ACTIVITY

OF URINE

Hoztrs

4 20 30 45

Group I (35S04=)

Group II (AA t 35S04=)

Group III (35S-AAS)

organic

inorganic

organic

inorganic

organic

1.9 13.6 15.8 16.3

36.2 66.3 80.8 84.2

3.7 11.9 15.1 16.2

20.1 65.6 79.7 83.7

69.5 83.4 84.8 87.2

in0 rganic 7.4 11.7 12.1 12.5

In

vivo SULFATION

TABLE

OF CHOLESTEROL

BY ASCORBIC

45

Z-SULFATE

3

DISTRIBUTION

OF 35S-ACTIVITY

Group

IN ADRENAL

GLANDS

Fraction ethyl ether d.p.m.lg

I (35SO4=) II (AA

ACID

t- 35S04’)

III (a%-AAS)

n-butanol

water %

d.p.rn./g

%

d.p.m./g

60

(1.3)

3498

10

(0.3)

2720

(78.1) (74.0)

926 945

10

(0.1)

6301

(73.9)

2213

% (20.6) (25.7) (26.0)

Adrenal glands

Of all animal tissues, adrenal glands contain the highest concentration of Lascorbic acid on a weight basis 36. Therefore, the adrenal glands from Groups I, II and III were homogenized and separated into three fractions: (a) ethyl ether soluble, (b) water soluble after barium chloride precipitation, and (c) n-butanol soluble. Table 3 shows the distributions of s5S-activity in each fraction. Surprisingly, all fractions were low in 35S-activity relative to the injected dose. The extracts from Group III rats contained approximately twice the s%-activity as the same fractions from the other groups of rats; however, the relative percentage distribution is nearly the same in all rats. CONCLUSION

We have found that when 3sS-AAS (20 mg per rat) was injected intracardially into rats, the fecal excretion of s5S-CS, relative to rats injected with s5S04=, was increased approximately 50 fold. In addition, AA (20 mg per rat) nearly doubled the excretion of s%-CS relative to Group I rats. Since radiochemical techniques were used, these data represent radiochemical comparisons and not necessarily molar comparisons. Also only a small number of rats were used and the samples were pooled eliminating any possible statistical treatment of the data. The injected s5S-AAS appears to be utilized unaltered and is not readily hydrolyzed in vivo within the time of the experiment. We are, therefore, proposing two possible explanations of these data (Fig. 3). The solid lines in Fig. 3 represent accepted reactions in biological sulfation. The broken lines represent the possible explanation of the involvement of AAS in this sulfation. AAS may be directly sulfating cholesterol (ROH) to form CS (ROSOaH) and this reaction could be highly specific. On the other hand, AAS may be reacting with 5’-AMP or 3’,5’-ADP to enhance the concentration of the normally accepted intermediates in biological sulfation (APS or PAPS). This would result in an increase in 35S-sulfation relative to %04=, not only with cholesterol, but perhaps with all sulfated intermediates, yielding a nonspecific sulfation. In view of the in vivo sulfate transfer from ssS-AAS to cholesterol resulting in

46

A. J.VERLANGIERI,R. 0. MUMMA

5’-ADP \

\ AA

Fig. 3. Possible sulfation mechanism involving L-ascorbic acid 2-sulfate. ROH represents cholesterol. ROSOaH represents cholesterol sulfate.

the increased excretion of ass-CS, as well as the identification of AAS as a naturally occurring compound, the observed hypocholesterolemic effects of AA could result in part from the natural biosynthesis of AAS from AA. This would be followed by the subsequent sulfation of cholesterol to CS which is then excreted in the feces. The quantitative significance of CS excretion relative to the total and the importance of the involvement of AA in biological sulfation

cholesterol pool needs additional

elucidation. ACKNOWLEDGMENTS

The authors Edward

McKee

would like to express their thanks for their technical

assistance

to Mr. Walter

Sapanski

and Mr.

in this study.

Note added in proof L-Ascorbic acid 2-sulfate has previously been reported as L-ascorbic acid 3-sulfate. However, recent X-ray studies have proven the structure to be L-ascorbic 2-sulfate [BOND, A. D., MCCLELLAND, B. W., EINSTEIN,J. R. AND FINAMORE, F. J., Arch. Biochem. Biophys., 153 (1972) 2071.

REFERENCES 1

MUMMA, R. O., AND VERLANGIERI,A.

as a possible explanation No. 2, 370 Abs. 2

J., In vivo sulfation of cholesterol by ascorbic acid 3-sulfate for the hypocholestemic effects of ascorbic acid, Fed. Proc., 29 (1971)

ZAITSEV, V. F., MYASNIKOV, L. A., KASATKINA, L. V.. LOBOVA, N. M., AND SIJKASOVA,T. I., The effect of ascorbic acid on experimental atherosclerosis,

Cor Vasa, 6 (1964) 19.

h

Vil’O SULFATION

OF CHOLESTEROL

BY ASCORBIC

3 GINTER, E., Effect of dietary cholesterol Med., 27 (1970) 23.

ACID Z-SULFATE

on vitamin C metabolism

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