treatment of “edible” soy protein, in gen- eral agreement with changes noted by De Groot and Slump,’ although more exten- sive. The greater destruction of methionine, serine, threonine, and arginine in the Wood- ard and Short experiment is probably re- lated to their more severe alkali treatment (pH 12 for eight hours at 60°C versus pH 12 for four hours at 40°C).

Thus, these two papers are in agreement in showing that alkali treatment of soy pro- tein is destructive of some amino acids and results in the formation of lysinoalanine. Woodard and Short did not comment on overall performance of their rats or on any physiological effects of the cytomegalia. It is unfortunate that neither of these groups of investigators has tested authentic lysino- alanine in otherwise complete diets. A dif-

ference between these papers relates to appearance of nephrocalcinosis in the rats of De Groot’s experiment, but not in those of Woodard’s and the cytomegalia of the pars recta in Woodard’s rats but not in those used by De Groot. As Woodard suggests, nephrocalcinosis might mask the presence of cytomegalia during histological examina- tion. In any event, these papers do serve as reminders that food-processing methods can have undesirable effects on nutritional quality and the wholesomeness of the prod- uct, and must be thoroughly evaluated be- fore being adopted on a wide scale. 0

1. A. P. De Groot and P. Slump, J. Nutrition 98:

2. J . C. Woodard and D. D. Short, J. Nutrition 45,1969

103: 569,1973

ASCORBIC ACID SULFATE (AAS): A METABOLITE OF ASCORBIC ACID WITH ANTISCORBUTIC ACTIVITY

Ascorbic acid sulfate has been identified as an important metabolite of ascorbic acid in man and several other species. The properties of this compound may explain at least

some of the functions of ascorbic acid.

Key Words: ascorbic acid, ascorbic acid sulfate, scurvy

The mode of action as well as the metabo- lism of ascorbic acid (AA) continue to be speculative. Apart from its serving as an antioxidant, there is some evidence to as- sociate ascorbic acid with certain hydroxy- lation reactions.

Another function of AA may lie in biologi- cal sulfation reactions. Mumma prepared ascorbic acid sulfate (AAS) and showed it to be an excellent sulfating agent in vitro and in vivo in the presence of a number of oxidizers, including mild oxidizers typical of biological systems. Based on this, Mum- ma and Verlangieri2 speculated that the anti-atherogenic effect of AA may be due to the formation of cholesterol sulfate, which is water soluble. Any compound facilitating its formation might facilitate cholesterol clearance and prevent its deposition in the arteries.

The natural occurrence of a compound having physical and chemical properties similar to AAS was first reported in the dry cysts of brine shrimp, artemia salina, by Mead and F i n a m ~ r e . ~ The compound was present in acid extracts of dry cysts. It was purified by chromatography on columns of Dowex-1 and DEAE cellulose and its homo- geneity established by paper chromatogra- phy in different solvent systems.

The authors felt that AAS may be a means by which the organism stores AA when en- cysted in the dry state. It would also be a repository of active sulfate.

More recently, Golub and Finamore have reported that while dormant embryos of artemia salina contain 2 to 3 pmoles of AAS per gram protein and no AA, during embry- onic development and emergence of larvae AAS disappears and AA appears in micro- molar quantities greater than AAS. The ori- gin of AA in artemia could not be traced to synthesis from other precursors like o-glu-

NUTRITION REVIEWSfVOL. 31fNO. BfAUGUST 1973 251

cose and D-glucuronic acid. Therefore the source of AA would be AAS or some other endogenous compound.

The presence of AAS in human urine has been reported by Baker et aL5 It was first detected in the urine of volunteers in whom scurvy had been induced by dietary means and who were given (l-14C, 4-3H) ascorbate. Urine from these subjects was fractionated into a lead acetate precipitate at pH 4 and pH 8 and a soluble fraction S. In each frac- tion a variety of metabolites containing 14C and 3H were detected. A qualitative correla- tion between the degree of scurvy and the percentage of l4C in the S fraction was ob- served in all the five subjects.

Electrophoresis of the S fraction revealed the presence of a major metabolite with a 14C to 3H ratio the same as that of the ad- ministered labeled ascorbate. The proper- ties suggested the metabolite to be a strong acid in which the carbon chain was almost certainly intact.

Its identity with AAS was established by co-ch romatog raphy with synthetically pre- pared AAS. The reference material used corresponded to the major monosulfate product of synthesis blocked at positions 5 and 6 and probable sulfate substitution at position 3. However, according to Mumma,6 recent x-ray studies have proven the struc- ture of AAS to be L AA, 2 sulfate.

A urinary metabolite having chromato- graphic and ultraviolet characteristics of authentic 35S-AAS could also be detected from the urine of normal male subject^.^ The S fraction corresponded to about 25 percent of the total metabolites of labeled ascorbate and in it the major radioactive component was AAS.5 Under conditions of high intake of AA (100 mg or more per day) much ingested ascorbate is excreted un- changed. The instability of free AA makes identification of true metabolites difficult. Only on a highly-restricted intake of ascor- bate is their identification possible. Besides human urine, this metabolite has also been found in the urine of guinea pig, rat, and trout and in tissues such as the liver and spleen of rats injected subcutaneously with

252 NUTRITION REVIEWSfVOL. 311NO. BfAUGUST 1973

35S042- and l-14C ascorbic acid.6 Thus it seems to be an ubiquitous metabolite.

AAS is stable at room temperature over a wide pH range,3 gives a positive methanolic FeC13 reaction,6 lacks the reducing property of AA,3 and on mild acid hydrolysis yields AA.3. 6 . Its ultraviolet absorption peak shifts from 230 nm at acid pH to 254 nm at alka- line pH. The corresponding maxima for AA are 245 and 265 nm respectively.

AAS has been shown to cure scorbutic symptoms in fish,' and very recently in guinea pigs.8 In the guinea pig study the biological activity of 2.5 mg of AAS (6.8 pmoles) was comparable to that of 1.5 mg AA (8.5 pmoles). Thus the compound was found to be at least as active as AA. High doses up to 100 mg per day did not affect growth or produce any pathological chang- es, suggesting its non-toxic nature.8

In vitro and in vivo studies in rats show that radioactivity from 35S AAS can be in- corporated into non-dialyzable insoluble membrane fractions. This may either be due to sulfation of protein or to intact incorpo- ration of the s t r ~ c t u r e . ~ Experiments with cultured human fibroblasts also suggest that AAS may be involved in sulfation re- actions.lo 0

1. R. 0. Murnrna, Biochim. Biophys. Acta. 165: 571, 1968

2. R. 0. Mumma and A. J. Verlangieri, fed. Proc. 30: 370, 1971

3. C. G. Mead and F. J. Finamore, Biochemistry 8: 2652,1969

4. A. L. Golub and F. J. Finamore, fed. Proc. 31: 706, 1972

5. E. M. Baker Ill, D. C. Hammer, S. C. March, 6. M. Tolbert, and J. E. Canham, Science 173: 826,1971

6. R. 0. Murnrna and A. J. Verlangieri, Biochim. Biophys. Acta. 273: 249, 1972

7. J. E. Halver, C. L. Johnson, R. R. Smith, 6. M. Tolbert, and E. M. Baker, Fed. Proc. 31: 705, 1972

8. R. 0. Murnma, E. E. McKee, A. J. Verlangieri, and G. P. Barron, Nutrition Rep. lnt. 6: 133, 1972

9. J. D. Carnpeau and S. C. March, Fed. Proc. 31 : 705,1972

10. A. D. Bond, fed. Proc. 31 : 706,1972