RESEARCH

Vitamins A and E help maintain lung health Vitamin Ε may protect vitamin A from destruction by air pollutants, while vitamin A directs formation of healthy cells in lining of the lung

The Battelle-Northwest chemists have conducted a series of nutrition experiments in which they fed rats a specially prepared diet that was high in polyunsaturate content. Some of the animals got food that was devoid of vitamin E. Others ate the same food supplemented with vitamin E.

When rats that weren't getting any of the vitamin were exposed to a stream of air containing 1 p.p.m. of ozone, they soon showed signs of se­vere pulmonary stress and died. In contrast, the animals who were getting the vitamin-supplemented food sur­vived twice as long in the ozone atmosphere.

In another series of experiments, rats were anesthetized after exposure

Vitamins appear to play a much more vital role in safeguarding lungs from the ravages of air pollutants than has been generally realized. For instance, it's entirely possible that vitamin Ε provides an important measure of pro­tection to vitamin A against destruc­tive oxidation by ozone and nitrogen dioxide, Battelle-Northwest's Dr. Jef­frey N. Roehm suggests. Vitamin A itself is essential for "steering" the dif­ferentiation process of epithelial cells toward formation of healthy, mucus-secreting tissue, MIT's Dr. Luigi M. De Luca told some 200 fellow sci­entists who attended a three-day sym­posium on pollution and lung biochem­istry that Battelle-Northwest hosted earlier this month at Richland, Wash.

Vitamin E, like related phenolic compounds, quenches free radicals such as those that are formed during the destructive degradation of the polyunsaturated lipid components of lung tissue by N 0 2 and 0 3 . This re­tarding of the rate of oxidative break­down is much more marked in the case of N 0 2 attack than it is for 0 3 , a fact that provides an important clue as to how these oxidizing agents differ from each other in the way they interact with long-chain unsaturated fatty acids, comments Dr. Daniel B. Men-zel, who heads Battelle-Northwest's nutrition and food technology re­search section in Richland.

In one series of experiments, Dr. Roehm, Dr. Menzel, and John G. Had-ley passed a stream of air containing from 1 to 5 p.p.m. of N 0 2 over sam­ples of methyl oleate, methyl linoleate, and methyl linolenate that were placed in small dishes within a glass cham­ber. In a separate series, 0 3 was used in place of N 0 2 .

Autoxidation. Nitrogen dioxide de­composes the polyunsaturated linole­ate and linolenate esters, but not the oleate. This provides strong evidence, the Battelle chemists maintain, that N 0 2 brings about autoxidation of poly­

unsaturates. The first likely step in the reaction sequence, they believe, is formation of a nitroxide free radical at one of the multiple double bonds fol­lowed by rapid autoxidation at methyl­ene carbon atoms. The greater the degree of unsaturation of the chain, in fact, the faster is the reaction rate, they note. But when free radical scavengers such as vitamin Ε are present, the onset of oxidation is de­layed.

Ozone, on the other hand, oxidizes both the mono- and polyunsaturates. Moreover, the effect of the presence of free radical scavengers isn't as marked as it is in the case of the N 0 2 experiments. What likely happens, Dr. Roehm conjectures, is that 0 3 forms an adduct across the olefinic double bond. This adduct splits to form a Creigi-type zwitterion. Some of tiiese zwitterions probably interact with water to form peroxide species that can cleave homolytically to yield free radicals. These, in turn, promote autoxidation of polyunsaturates.

Malonaldehyde is one of numerous products formed when N 0 2 and 0 8 decompose the unsaturated fatty acid model compounds. Dr. Roehm and his coworkers use the buildup of mal­onaldehyde concentration as an in­dicator of the rate of decomposition. They do this by noting the intensity of the color that develops when 2-thio-barbituric acid, which forms a com­plex with malonaldehyde, is added to samples withdrawn from the reaction chamber at different time intervals.

The rate of malonaldehyde forma­tion is very high. More than 1000 p.p.m. results when methyl linolen­ate is exposed to 1.5 p.p.m. of N 0 2 or Oa for two hours.

Malonaldehyde is a very undesirable material to have in a biological sys­tem, Dr. Roehm points out, because of its considerable degree of reactiv­ity. It cross-links DNA, and it de­activates many enzymes, he notes.

Dr. Daniel B. Menzel removes rat from chamber after exposure to ozone

to 10 p.p.m. of N 0 2 in their air supply. Their lungs were removed, washed, and homogenized in a solution of chloroform and methanol to extract the lipids. Following esterification, the fatty acids were separated by gas chromatography. The animals that had eaten the vitamin Ε-deficient diet had a significantly lower level of poly­unsaturated compounds in their lung tissue than was the case for animals that received the vitamin in their food.

Besides playing a primary role in protecting lung tissue lipids from oxi­dative degradation by N 0 2 and 0 8 , Dr. Menzel thinks that vitamin Ε also might safeguard the conjugated iso-prenoid side chain of vitamin A against destruction by these oxidiz­ing air pollutants. This in itself would be an important function, be­cause it is now becoming increasingly evident that vitamin A is crucial for the healthy metabolism and growth of epithelial cells.

Recent experiments at MIT's de-

38 C&EN JUNE 29, 1970

partment of nutrition and food science, for example, have shown that vita­min A influences the biochemistry of glycoprotein synthesis in lung tissue. Absence of the vitamin from the diet of experimental animals results in sub­tle differences in the composition of the glycopeptides, Dr. De Luca notes. This, in turn, might be linked to the generation of thick layers of keratin­izing squamous cells in the bronchial mucus during times of vitamin A de­ficiency, he adds. Epithelial stem cells give rise to these squamous cells instead of normal, mucus-secreting ones, he notes, if the vitamin is ab­sent from the diet.

Dr. De Luca and his coworkers, Dr. G. Harvey Anderson and Dr. George

Wolf, use 14C-glucosamine as a tracer in their studies of glycopeptide syn­thesis by lung tissue. Following in­cubation of tissue slices in the pres­ence of the radioactive marker com­pound, they digest the tissue with pro­teolytic enzymes, precipitate the glyco­peptides with ethanol, and separate them by stepwise elution from a Sephadex column using lithium chlo­ride solutions of increasing strength.

The characteristics of the glycopep­tides recovered from tissue grown in the presence or absence of vitamin A, as well as the rate at which they are synthesized, are identical except for one fraction which is made much more slowly in cases of vitamin A-de-ficient animals. When the animals are

up again, and the cell differentiation process reverts to formation of healthy tissue, Dr. De Luca points out.

These findings raise a number of in­triguing questions. It's obvious, he notes, that the fucose-rich glycopep­tide is implicated in some way in the process whereby a cell undergoes dif­ferentiation to form healthy, mucus-secreting tissue. "What we don't know yet is whether vitamin A fosters the biosynthesis of the glycopeptide which in turn acts on the mucus cell differentiation mechanism, or whether the vitamin stimulates cell differentia­tion directly, with the fucose-glycopep-tide being a product of the differentia­tion process/' Dr. De Luca says.

Another question that the MIT bio-

Dr. G. Harvey Anderson (left), Dr. Luigi M. De Luca, and M. Schumacher are studying effects of vitamin A on tumor cells transplanted into hamsters

Dr. Jeffrey N. Roehm finds malonalde-hyde from oxidation of fatty acids

fed the vitamin, the rate of biosynthe­sis of this glycopeptide is restored to normal levels within 18 hours.

Fucose-glycopeptide. This partic­ular glycopeptide fraction recovered from hamster lung tissue appears to be a common constituent of mucosa tissues in general, Dr. De Luca ob­serves. For instance, it is very similar in chemical composition and physical properties to a glycopeptide he iso­lated from the intestinal mucosa of rats. That glycopeptide contains glu­cosamine, galactose, fucose, and sialic acid, he and his coworkers have found. Ultracentrifugation in a phosphate buffer shows that it comprises two components with sedimentation con­stants of 6.2S and 7.2S.

At the same time that formation of the fucose-containing glycopeptide slows down, the tissue tends to be­come keratinized (rich in fibrous pro­teins) instead of mucus-secreting. But when vitamin A is reintroduced to the diet, the rate of synthesis picks

chemists are asking is whether excess vitamin A might reverse the progress of a keratinizing tumor such as bron­chial cell carcinoma. To help find the answer they are collaborating with the National Cancer Institute's Dr. Um­berto Saffiotti, who has shown that high doses of vitamin A, given orally to benzpyrene-treated hamsters, can inhibit completely the appearance of squamous tumors of the lung.

To study the effect of excess vita­min A on a benzpyrene-induced ana­plastic tumor of hamster lung, the MIT researchers transplant the tumor cells into healthy hamsters—some of which have been given large doses of vitamin A for 12 days prior to the transplant. The tumor grown in vitamin A-treated animals shows an increased ability to incorporate 14C-glucosamine into gly­copeptide. Whether an increase in the glycopeptide level of the tumors might effect a change in cell structure and tumor growth rate still remains to be seen, Dr. De Luca adds.

JUNE 29, 1970 C&EN 39