Vitamin B12 결핍에 의한 백서 설점막의 형태학적 변화에 관한 연구

Abstract

[한글]

구강점막상피는 체내에서 세포의 성장과 교체시간이 가장 빠른 조직의 일종으로서 전신적 건강상태에 따라 민감한 반응을 보이며 특히 영양결핍증의 경우에 심한 영향을 받게된다. 영양결핍증중에서도 수용성 vitamin의 부족은 구강내에 구각염, 치은출혈, 위축성 설

염 및 재발성 궤양 등을 초래한다. 구각염은 riboflavin결핍의 특징적인 소견이며 vitamin B^^12, 엽산 및 철분결핍은 설점막 사상유두의 위축을 초래한다.

Vitamin B^^12 (B^^12) 는 cobalt와 phosphorus를 포함하는 결정형 복합체로 coenzyme으로서의 기능을 가지며 purine합성과 thymidylate형성에 관여하게 된다. 따라서 B^^12 가 부족하게 되면 DNA합성에 장애를 일으키게 되어 megaloblastic anemia, 위장관증상,

위축성 설염 등을 초래하며 척수와 뇌에 수초탈락도 볼 수 있다.

그러나 실험적으로 B^^12 결핍을 일으켰을때 형태학적 변화는 유도하기 힘들며 더구나 설점막에 관한 관찰은 보고된 바 없다.

이에 저자는 정상 혹은 간부분절제를 한 50∼80gm의 자성백서를 B^^12 결핍사료로 4개월간 사육하여 매 1개월마다 일부를 도살하여 혈청내 B^^12 량을 측정하였고 말초혈액과 골수의 도말표본을 검색하고, 설점막을 광학 및 전자현미경적으로 관찰하여 다음과 같은

결과를 얻었다.

1. 정상백서를 B^^12 결핍사료로 4개월간 사육하면 1개월후부터 혈청내 B^^12 치가 감소하기 시작하여 4개월째에는 약 1/3까지 감소되었고 간장을 부분절제하면 4개월째에 1/4이하로 현저히 감소되었다. 그러나 혈청내 엽산과 철분양에는 변동이 없었다.

2. B^^12 결핍사료로 4개월간 사육하여도 말초혈액의 혈색소, Hct, 평균적혈구용적 등에는 변동을 일으키지 않았으며 중성백혈구의 과다분절현상이 3개월째부터 관찰되었다.

3. 골수는 B^^12 결핍사료로 사육한 3개월째부터 적혈구계 증식과 세포분열 및 거대한 metamyelocye가 관찰되었으나 거대적아구성증식은 일어나지 않았다.

4. 설점막의 조직소견상 B^^12 결핍사료로 사육한 군에서는 기저세포 핵의 증대가 관찰되었고 많은 세로분열상과 기저층의 증식이 관찰되었으며, B^^12 결핍이 지속되면 착각화현상이 일어나고 간부분절제군에서는 상피층의 위축현상도 일어났다.

5. 전자현미경 소견으로 전 실험군에서 상피세포의 세포질내에 mitochondria의 증가 및 종창이 관찰되었고 핵막이 심히 불규칙해졌다.

이상의 소견으로 보아 B^^12 결핍사료로 4개월간 사육하면 혈청내 B^^12 량은 급격히 감소하나 megaloblastic anemia현상은 일어나지 않고, 다만 과립구계통의 성숙장애 및 설점막의 위축과 상피세포 성숙부전현상을 초래함으로 보아 B^^12 결핍때에 설점막 변화가

악성빈혈현상에 선행하는 것으로 사료된다.

[영문]

Oral mucosa, representing a rapid cell growth and turnover rate, is regarded as an indicator reflecting systemic conditions, and is vulnerable to nutritional deficiency (Dreizen, 1972). In particular, deficiency of water soluble vitamin induces cheilosis, gingival bleeding, atrophic glossitia and recurrent ulcerations.

Characteristically atrophic glossitis results in deficiencies of vitamin B^^12, folic acid and iron.

Vitamin B^^12 as a crystalline compound containing cobalt and phosphorus, has a major function as a coenzyme and is associated with purine synthesis and thymidylate formation(Harper et al,, 1979). Therefore B^^12 deficiency interferes with DNA synthesis resulting in megaloblastic anemia, gastrointestinal symptoms,

atrophic glossitis, demyelinigation of the spinal cord and brain, and axonal degeneration (Robbins and Cotran, 1979).

However, in experimentally induced B^^12 deficiency, no tissue injury is demonstrable in spite of presence of biochemical dysfunction such as methylmalonic aciduria(Frenkel and White, 1973: Kark, 1974: Siddons, 1974). Furthermore, the morphological study about the tongue mucosa has not been studied experimentally.

Therefore the present experiment is undertaken to investigate the morphological changes of the tongue mucosa in B^^12 deficiency by light and electron microscopic observations.

Materials and Methods

Female albino rats weighing 50∼80 gm were used for the experiment, and divided into four groups as follows:

Group Ⅰ: Normal diet 16 rats

Group Ⅱ: Vitamin B^^12 deficient diet 16 rats

Grous Ⅲ: Normal diet after hepatectomy 20 rats

Group Ⅳ: Vitamin B^^12 deficient diet after hepatectomy 20 rats

The experimental diet was prepared by the recipe according to Frenkel and White(1973).

Partial hepatectomy was performed in order to reduce the amount of B^^12 storage in the liver. The median, left and caudate lobes were removed by the method according to Higgins and Anderson(1931).

The rats were sacrificed at one month intervals for four months and fresh blood, bone marrow and tongue tissue were obtained.

Serum B^^12, folic acid and iron values were determined from fresh blood. Determination of serum B^^12 and folic acid were performed by radioimmunoassy, and serum iron was determined by Hyland's Ferro-chek Ⅱ test.

Hemoglobin, hematocrit and mean corpuscular volume were measured from blood in EDTA tubes. Peripheral blood smears were examined by Wright staining, and bone marrow aspiration smears obtained from femur were examined by Wright-Giemsa staining.

For histological observation of the tongue mucosa, routine hematoxylin-eosin staining was employed.

Fer electron microscopic observation, the tongue tissue was fixed in 3% glutaraldehyde as well as in 1% osmium tetraoxide(in phosphate buffer pH 7.4); a routine upon embedding procedure was applied by uranyl acetate and lead citrate staining. The observation was made with a Hitachi H-500 model electron microscope.

Results and Summary

1. In the B^^12 deficient group serum B^^12 value began to decrease after one month and was reduced to approximately one third of normal value at four months. The value of the hepatectomized B^^12 deficient group was reduced to one fourth. Serum values of folic acid and iron did not change.

2. In both simple B^^12 deficient and hepatectomized B^^12 deficient groups, hemoglobin, hematocrit and mean corpuscular volume remained in the normal range, hypersegmentation of neutrophils was observed at the third and fourth months.

3. In both B^^12 deficient groups the bone marrow showed hyperplasis of erythroid series with many mitotic figures without megaloblastic change, but giant metamyelocytes were observed during the third and fourth months.

4. In light microscopic observations the atrophic changes of the tongue mucosa were detected only in the hepatectomized B^^12 deficient group. In both B^^12 deficient groups basal cell hyperplasia wag observed with increased mitotic figures, and vacular degeneration with parakeratosis was also noted. The nuclei of basal cells were markedly enlarged from the first month of the experimental period.

5. Electron microscopic observations revealed increased number of mitochondria in the cytoplasm of basal cells, pronounced nuclear infolding and marked chromatin clumping in B^^12 deficient groups.

In summary, results obtained by the present investigation suggest that megaloblastic anemia does not develop with a four month period of B^^12 deficient diet, although serum B^^12 decrease markedly. On the other hand mucosal changes of

the tongue develops, indicating that tongue changes precede the development of megaloblastic anemia in B^^12 deficiency.