Keratomalacia

Most of the dietary vitamin A is derived from plant foods in the form of pro-vitamin A, the carotenoids. Though in 1930 it was first demonstrated that beta-carotene is the precursor for vitamin A and it is well accepted that I mole of beta-carotene is equivalent to one mole of vitamin A, the mechanism of conversion to vitamin A has been controversial. Some of the mechanisms suggested are central cleavage potentially yielding 2 molecules of vitamin A or excentric cleavage producing one molecule of vitamin A from beta-carotene which drastically varied the potency of carotene. A mucosal supernatant from rat intestine was shown to have beta-carotene dioxygenase activity which provided the basis for central cleavage. Many observations on enzyme activity in vitro and efficacy of carotene in vivo did not support the above findings and a re-evaluation of the whole problem was undertaken at the National Institute of Nutrition (NIN), Hyderabad. Intestinal conversion of beta-carotene to vitamin A both in vitro and in vivo in rats and in vivo in children was evaluated. A novel method of obtaining the in vivo conversion of carotene to vitamin A using the ratio of area under plasma vitamin A time curves after a dose of beta-carotene and vitamin A ( >100 (mu)g) was developed in rats and later extended to children. In children a dose of 1.5 mg of beta-carotene and vitamin A was used. From these studies intestinal conversion of beta-carotene to vitamin A was found to be an enzymatic reaction involving central cleavage and which needed the presence of oxygen. The substrate was found to bind the enzyme at C-15,15'. The enzyme may be associated with inherent or contaminant enzyme which breaks the other part of the molecule released after central cleavage of carotene. The in vivo conversion of carotene to vitamin A was found to vary from 20 to 80 per cent depending on the nutritional status. Vitamin A deficiency was found to enhance both the in vitro and in vivo conversion and protein deficiency to decrease both. Thus the present results confirm the convertibility of dietary carotenoids to vitamin A and could facilitate further investigations on interactions of different dietary carotenoids on the absorption and cleavage of carotene to vitamin A in children.

Key words beta-carotene - children - in vitro enzyme activity - in vivo conversion - rats - vitamin A conversion

Vitamin A, also known as all trans retinol, is essential for normal vision and for maintaining the integrity of epithelial cells. Besides, it is needed for a wide variety of physiological functions. Deficiency of vitamin A results in impaired dark adaptation and leads to night blindness as the early manifestation of deficiency, followed by conjunctival xerosis and Bitot spots. Corneal xerosis, keratomalacia and total blindness are manifestations of severe vitamin A deficiency. The non-ocular manifestations include impaired growth, altered immune response, and reproductive disturbances, primarily, in animals. Recent studies show that vitamin A deficiency is associated with increased morbidity and mortality in children probably through impairment in specific and non-specific immune mechanisms1.

Vitamin A deficiency is widespread in India and the neighbouring countries of South-East Asia where serum vitamin A levels in population groups are below 20 (mu)g/dl. The daily intake of vitamin A in such populations ranges fr.em 100-300 (mu)g of retinol (retinol equivalent; RE), most of it derived from vegetable sources2 In ri,ral India, the intake of vitamin A is around 100 RE in children and 250 RE in adults. On the other hand, in countries where vitamin A deficiency signs are seldom seen, serum levels of vitamin A are above 30 (mu)g/dl. Similarly, in high income groups in our country which rarely develop deficiency, the daily intake of vitamin A in adults ranges from 400-500 (mu)g2,3. A prophylactic measure consisting of a six-monthly oral dose of 200,000 IU of vitamin A as supplement has been developed by NIN as a short term public health programme in children of 2-5 yr age. This programme has been in operation for the last 27 yr and covers all the states of the country and its efficacy has been well documented4. However, in the long run, food-based approaches have been advocated for control of vitamin A deficiency which have many advantages, they are self-sustainable and increase the intake of other nutrients simultaneously. Dark green leafy vegetables and yellow and orange fruits, which are good sources of pro-vitamin A carotenoids, especially beta-carotene, are mostly promoted as means of improving vitamin A status5.

Structural requirements

Chemically vitamin A contains a beta-ionone ring and a side chain of I 1 carbons with 4 alternate double bonds and these are necessary for biological activity. This requirement makes the carotenoids possessing similar chemical groups only as the precursors or pro-vitamin A compounds. By appropriate oxidation at the central carbon 15, 15', beta-carotene can form 2 molecules of retinal (vitamin A aldehyde) while other pro-vitamins get converted to one molecule of retinal (Fig. 1). Retinal is reduced to retinol (vitamin A alcohol) in the body1.

Dietary sources : Foods provide vitamin A either in the preformed state from animal foods such as milk, butter, egg and fish or from its precursor carotenoids, especially beta-carotene, derived mainly from leafy vegetables and yellow and orange coloured fruits. Recent data6 on carotene content of common Indian foods is presented in Table I. beta-carotene and other carotenoids are converted to vitamin A in the intestinal cells. After absorption, retinol is esterified and the amount in excess of immediate requirement is stored in the liver. On demand, retinol from the liver is released attached to the carrier protein or retinol binding protein (RBP) and is transported to the target tissues1.

The bioavailability of nutrients is related to the efficacy with which the nutrients present in the food are utilised for performing biological functions. This includes (i) release of nutrients into the gut lumen from the food matrix following digestion; (ii) the uptake by mucosal cell and their transport into the body; and (iii) wherever applicable conversion of the precursor to the active ingredient in the mucosal cell. All three stages are involved in the bioavailability of pro-vitamin A compounds from food.

As far as carotenoids present in plant foods are concerned, several factors like the rate of release from the food particles, size of the food particles, dietary fibre, dietary fat and efficiency of digestive enzymes can facilitate or inhibit their biological utilisation. Since carotenoids are lipid-soluble they have to be emulsified to form mixed micellae in which pancreatic lipase and bile salts from the liver allow interaction of carotenoids with mucosal surface. Heating of plant food before ingestion improves the bioavailability of carotenoids and may also lead to destruction of some carotenoids by oxidation. Presence of antioxidants can protect food intake from destruction. Carotenoids also can be lost due to interaction with insoluble substances such as dietary fiber. There can also be competitive interactions between and among carotenoids7.

Absorption of carotenoids

Absorption, metabolism and transport of carotenoids have been reviewed recently by Parkers and Furr and Clark9. Relatively little is known quantitative ly of the efficiency of intestinal absorption of pro-vitamin A carotenoids such as alpha, beta-, and gamma-- carotenes and cryptoxanthin.

In humans, approximately 8-17 per cent of orally administered labelled beta-carotene is absorbed via the lymphatic system and 60-70 per cent of the absorbed radioactivity is found as retinyl esters, whereas IS per cent remains as intact beta-carotene10.

Sheffield experiments carried out in human subjects indicated that 1 (mu)g of retinol is equivalent to 2.5 (mu)g of absorbed beta-carotene and studies in USA" have shown that the requirement of beta-carotene is approximately twice that of retinol. These studies dealing with human experiments formed the basis for fixing the efficiency of beta-carotene conversion to vitamin A as 50 per cent.

The Joint FAO Expert Group12 reviewed the published studies and reported a wide range of values for absorption, the mean value of which worked out to 33 per cent. Assuming 50 per cent intestinal conversion, the suggested conversion factor for one (mu)g beta-carotene was 0.167 (mu)g retinol and it is widely accepted. Adopting similar techniques mainly with green leafy vegetables and papaya fruits and carrots in India the absorption was found to vary between 35 and 81 per cent with a mean value of 50 per cent. The Expert Group of ICMR'3 therefore recommended 50 per cent absorption from Indian diets containing carotene rich plant foods, this aspect has been recently reviewed7. Thus the recommended conversion factor is that 1.0 (mu)g of beta-carotene is equivalent to 0.25 (mu)g of retinol.

In most animals like rat, rabbit, chick and pig dietary beta-carotene is converted to retinoids in the small intestine without significant absorption of intact beta-carotene. On the other hand in humans, significant amounts of beta-carotene are absorbed as such into the body14. Recently, it was found that ferrets15 and gerbils16 are suitable animal models for studying the absorption of carotenoids which is similar to that of humans. The absorption of beta-carotene is influenced by other carotenoids. In ferrets, canthaxanthin or lycopene reduced the 0-24 h plasma beta-carotene response when given concurrently, compared to administration of beta-carotene alone17. Similarly, in humans, the plasma beta-carotene concentration response was reduced by 40 per cent when 25 mg of canthaxanthin was given along with 25 mg of betacarotene18. When a combined oral dose of lutein (15 mg) and beta-carotene was given there was a 40 per cent reduction in plasma lutein response in humans19. But lutein has no effect on beta-carotene absorption. The mechanism of this interaction is not known.

Conversion of carotenoids to vitamin A

Among all the carotenoids beta-carotene is the most potent precursor of vitamin A. Apart from the conclusive evidence that beta-carotene serves as a precursor, the mechanism of conversion of beta-carotene to vitamin A remains as a subject of controversy.

Several authors independently demonstrated that when beta-carotene was given tc vitamin A deficient rats orally, considerable amount of vitamin A was found in their livers. They concluded that the intestine is the major site of conversion, not the liver. beta-carotene conversion was also demonstrated using rat intestinal loops and isolated rat liver14.

A cell free conversion of beta-carotene by enzymes isolated from homogenates of rat intestinal mucosa and later from livers was demonstrated20. The enzyme is termed as 'beta-carotene dioxygenase' (EC .13.11.21). Later many investigators preferred the term `carotene cleavage enzyme'1. Requirement of an appropriate detergent-lipid combination like sodium glycocholate and egg lecithin was found necessary for the optimum reaction to take place. Carotene cleavage activity was later demonstrated in the intestines of several species like guineapig, rabbit, tortoise, monkey, fresh water fish and chicken but not in the cat21.

Mechanism of conversion of beta-carotene to vitamin A

Two major oxidative pathways for the conversion of beta-carotene were postulated by Glover in 1960(22): (i) the cleavage at the central double bond to yield two molecules of all trans retinal, and (ii) step-wise excentric cleavage to yield a sequential set of betaapocarotenals which ultimately is converted to one molecule of all trans retinal and a variety of smaller fragments. However, controversy still exists between central or excentric cleavage of beta-carotene.

Comparative growth promoting activities have consistently shown that oc- and gamma-carotene are only half as active as 3-carotene supporting the idea that the carotene molecule is cleaved centrally. For many years there has been general agreement on two structural requirements for pro-vitamin A activity of carotenoids. One demands the presence of unsubstituted beta-ionone ring and the other, an unaltered side chain with double bonds1. But the poor in vivo vitamin A potency of 3-carotene when compared to pure vitamin A raised doubts about the central cleavage of beta-carotene14,22. Even on physicochemical grounds it was suggested that because of resonance, the centrally located double bond of a conjugated system will be more stable than the terminal ones which would imply that terminal attack is preferred to central fission.

After reviewing the situation as it existed in 1960, Glover22 favoured the beta (-oxidation theory' based on his experimental evidence of the deposition of 15'-^sup 14^ Cretinol in the livers and small amounts of labelled 12'-apo-carotenal and its derivatives in the intestines of vitamin A deficient rats when fed with a mixture of labelled beta-carotene and unlabelled 12'-apo-betacarotenal. According to this theory, the oxidative cleavage starts at either end of the ethylenic chain with equal probability. The oxidative cleavage then continues with successive removal of two carbon units until C^sub 20^ unit is reached. Further oxidation is blocked by the methyl group located on the C^sub 13^, which is at the beta-position with respect to the central carbon atom of the molecule. This is depicted in Fig.

1. Ganguly and Sastry23 reviewed the evidences for intestinal conversion of beta-carotene and challenged the central cleavage mechanism based on the observation that beta-apo-carotenals were isolated in vivo from chicken intestine.

Disconcerting reports on carotene conversion

In order to decide which of the mechanisms is operative (central vs random) using new techniques like HPLC, Hansen and Maret24 questioned the enzymatic conversion of beta-carotene to vitamin A by a dioxygenase. Wang et al25 have shown strong evidence in support of enzymatic excentric cleavage of beta-carotene.

The chemical instability of carotenoids when exposed to O^sub 2^ complicates the study of beta-carotene metabolism. Many products of the autoxidation of beta-carotene are similar to those thought to be produced via enzymatic means. Such products include retinal, beta-apo -10',-12'- and 14'-carotenals.

Many observations on enzyme activity in vitro and efficacy of carotene utilisation in vivo did not support these findings and a re-evaluation of the whole problem was called for26.

Lack of improvement in vitamin A status in Indonesian women who were fed for prolonged periods with green leafy vegetables compared to controls who consumed wafers containing a similar amount of beta-carotene showed the effect of food matrix on the availability of carotenes27. A similar insensitivity of plasma vitamin A to even synthetic beta-carotene along with food carotene in Guatemala children raised questions on the efficiency of betacarotene conversion to vitamin A28. These studies came at a time when there was a global consensus that control of vitamin A deficiency should be through horticultural or food-based interventions.

Solomons and Bulux29 discussed the evidence available and concluded that the 'patchwork' of current evidence and theoretical speculations raises doubts about correcting vitamin A deficiency exclusively through a horticultural strategy and stressed the inevitable role of supplementary vitamin A in developing countries. They called for new research using stable isotope technology to resolve the various issues.

de Pee et al 30 have recently reviewed the literature on the prospects of combating vitamin A deficiency through food based approaches. Cross sectional studies reported the rise in the plasma levels of both beta-carotene and retinol with consumption of carotene- containing foods though in some studies the increase in retinol was not correlated with that of carotene. This may be interpreted to reflect concurrent intake of carotene and retinol rich foods in the population rather than the conversion of carotenoids to retinol. While case-control and community studies provided useful data in suggesting the association between consumption of carotene-- rich foods and improvement in vitamin A status, the issue needed more careful examination. Early intervention studies in Europe had shown the effectiveness of carotene from vegetable sources like peas, spinach and carrots on vitamin A status.

Many reports from developing countries listed by de Pee et al30 were stated to suffer from design defects like lack of control groups and base-line information, small sample size, unexplained drop out rate and absence of complete dietary information. The authors focused on three studies which fulfilled their criteria of including negative control group and sufficient numbers. One study reported increase in serum retinol after feeding sweet potatoes and dark green leafy vegetables for 24 days. The second study27 found no improvement in vitamin A levels after feeding green leafy vegetables over 12 wk while there was an improvement after consumption of betacarotene-fortified wafers. The third study28 was conducted in children after vitamin A repletion and there was no increase in serum retinol after feeding carrots along with fat.

Serum retinol levels were found to be significantly higher after administration of red palm oil or after consumption of 'buruti' sweet which is derived from palm fruit. It is concluded that the state of carotene whether it is pure and free, or it is bound to the surrounding structures of the food matrix is important for the availability and conversion29.

Narasinga Rao7 has recently reviewed the literature from the Indian subcontinent and concluded that the evidence strongly favours the conversion of carotenoids to vitamin A. Critical examination of the controversial reports, revealed that the dietary carotenoids were effective in increasing the plasma vitamin A in the population where plasma vitamin A levels were lower and not in those in whom plasma vitamin A levels were higher. In the later study Bulux et al 31 confirmed the inverse relationship between initial plasma vitamin A and the rise in it after dietary carotenoids. This explained why some of the earlier observations did not reveal the effectiveness of dietary carotenoids.

Subsequently, Furr and Clark9 examined the concept of classifying subjects who display large increases in serum beta-carotene concentration after dosing (respondents) and others who showed only little or slight increase (non-respondents). The evidence from different studies did not favour the existence of non-respondents.

Keeping many of the discordant observations in view, Wolf in 1995(32) reviewed the conversion of betacarotene and concluded that `the enzymatic cleavage of beta-carotene: still controversial'. Thus, the controversy in carotene conversion lies about the quantitative and qualitative aspects of not only carotene absorption from dietary sources but also the generation of vitamin A from beta-carotene.

A re-evaluation of the issues: Studies at NIN

It appeared essential, therefore, to re-examine the various issues involved in carotene conversion both in vitro and in vivo under the same set of experimental conditions, using standard procedures of analysis. To achieve these objectives both in vitro and in vivo experiments were planned in rats. de Pee et al30 have called for vigorous experimental studies to prove the issue related to pro-vitamin A activity of dietary carotenoids. The possible effect of alterations in the intestinal enzyme activity brought about by nutritional deficiencies accounting to variations in in vivo conversion has also been investigated in the present studies at NIN33.

SUMMARY OF NIN STUDIES

In vitro studies

The results in brief confirm that the intestinal conversion of beta-carotene to vitamin A in rats is an enzymatic reaction involving central cleavage and needs the presence of oxygen.

As in the earlier reports20,21,24, various methods of purification of the enzyme like differential centrifugation, salting out and gel filtration have been tried on the rat intestine. The enzyme products were analyzed by using HPLC methodology. The efforts of purification were not successful. The enzyme, at best, was enriched 2-3 fold in the retentate when the intestinal supernatants were ultrafiltered through 100 kDa filter. The size of the enzyme seems to be greater than 100 kDa. Even in the most enriched state, the enzyme unusually has over 1000 fold poorer specific activity (10-13 pmoles/h/mg protein) as compared to other enzymes (usually micromoles). This low specific activity renders the enzyme more difficult to purify.

The optimum conditions of substrate concentration (k^sub m^= 0.5 (mu)M), pH (7.7) and temperature (37 deg C) have been confirmed with the rat intestinal enzyme. For maximal activity glycocholate (detergent) and egg lecithin (phospholipid) were found necessary, as referred earlier20, probably to facilitate solubilization of carotenoid substrates in the micellar phase. The activity was. better in the presence of nicotinamide and reduced glutathione. Incubation of the enzyme source with different substrates and even with different apocarotenals at optimum concentrations (5 times the k^sub m^ value of beta-carotene), using different buffers did. not reveal any product other than all trans retinal confirming the central cleavage. The competitive inhibition obtained with 15-15' dehydro 10' beta-apocarotenal (DAC) with a triple bond replacing the usual double bond between carbon 15 and 15' demonstrated that the central carbon atoms are involved both in binding with the substrate site of the enzyme and in the central cleavage to form the retinal (Table II).

All the substrates tested had all trans configuration and unaltered side chains. The conclusions from these data are that the enzyme has stringent structural requirement about the ring, as has been generally believed. The enzyme tolerated no modifications in structure except a single ring hydroxylation as in beta cryptoxanthin. This substrate showed enhanced enzyme activity even in the presence of optimal concentration of beta-carotene. It is imperative that betacryptoxanthin should yield one molecule of retinal and another of 3-hydroxy retinal as products after central cleavage by the enzyme. In analogy, incubation with alpha-carotene as substrate should result in the formation of alpha-retinal along with retinal. However, no second product, apart from retinal, was detected when cryptoxanthin was used as a substrate. But with alpha-carotene, a second peak corresponding to alpha-retinal was found eluting with beta-retinal. The enzyme source is perhaps endowed with additional activity to further degrade the second molecule (3hydroxy beta-retinal) with hydroxy substitution in the ring that is released. However, the activity does not seem to act on alpha-ionone ring (in which the double bond is out of conjugation), thereby the product released in the first reaction, alpha-retinal, is not degraded. This explains why in normal circumstances the second molecule of retinal that is released even from beta-carotene is not available due to degradation due to the presence of second enzyme activity. This will also explain why the retinal formed with alphacarotene as substrate is 90 per cent of that with betacarotene under the described experimental conditions.

Canthaxanthin with both rings substituted by keto groups is not accepted as substrate and does not allow interaction of the enzyme with beta-carotene. But if one of the groups is further hydroxylated as in zeaxanthin and astaxanthin they do not interfere, at least, with the oxidation of beta-carotene but by themselves they are not substrates. Thus hydroxylation of the second ring moiety of carotenoid seems to stimulate the beta-carotene cleavage and confirms the data obtained with cryptoxanthin.

Carotene dioxygenase activity may be thus associated with an inherent, or contaminant enzyme activity, which breaks the other part of the molecule containing modified groups resulting in the release of only one mole retinal.

In vivo studies

One of the other highlights of the study is the development of a simple potential method of obtaining in vivo conversion of synthetic beta-carotene to vitamin A in rats and extending the same to children. The method is based on pharmacokinetic principles and uses the ratio of area under the plasma concentration-time curves of vitamin A obtained after a dose of beta-carotene as well as preformed vitamin A, as a measure of in vivo conversion.

In the initial experiments, different doses of beta-- carotene and vitamin A were administered to rats and the plasma levels were determined at different time points. To obtain reliable increase in plasma vitamin A area-under curves (AUCs), it was necessary to draw blood samples at 0, 4, 8 and 12 h after the doses. The AUCs for vitamin A were found to be linear with different doses of both beta-carotene and vitamin A in rats above the doses of 100 (mu)g. The slope ratios reflected the overall ratios of AUCs in the entire dose range. The ratio varied between 0.20 to 0.86 in different groups of rats depending on their plasma vitamin A concentration. The higher the initial plasma vitamin A the greater was the ratio. For example, rats with a plasma vitamin A of 35 (mu)g/ dl had a ratio of 0.86 and those with 18 (mu)g had a ratio of 0.20. Under similar conditions, Fig. 2 shows the pattern of plasma vitamin A profile after a dose of 1.5 mg of beta-carotene and vitamin A in children. The mean value for conversion obtained from a set of six children varied between 0.21 - 0.32 and is comparable to that obtained in rats. Other studies from NIN34,35 using natural sources of beta-carotene like red palm oil and green alga, spirulina, have shown conclusively that dietary carotenoids are converted to vitamin A.

This method can facilitate further studies on food matrix and interaction of different dietary carotenoids on the absorption and cleavage of other carotenoids in children to identify promoters and inhibitors of carotenoids for dietary counselling.

The effect of altered nutritional status like vitamin A deficiency and protein deficiency on in vitro conversion as well as in vivo conversion of beta-carotene were also studied (Table III). In vitamin A deficient rats the enzyme activity was found to be significantly higher (P

Based on these studies one could identify and advocate the use of certain promoters or avoid the use of inhibitor elements in food. For example, consumption of mango has been shown by Pingle and Sivakumar38 to improve both vitamin A status and iron status in tribal children. Mango contains considerable amounts of beta-cryptoxanthin along with beta-carotene and was shown to enhance in vitro conversion to vitamin A in the present studies. On the other hand, 2 yr after horticultural intervention through home gardens and bringing about an increase in consumption of green leafy vegetables, no improvement in vitamin A status was observed in rural children39. With these developments, the challenge to eliminate vitamin A deficiency in the next 10-15 yr seems to be achievable only if proper dietary factors are considered in nutritional education programmes.

While the horticultural approach through home-- gardening or through food-based intervention had been declared as the preferred means of controlling vitamin A deficiency at the national and global levels5, it is essential to intensify the following actions7:

i. Identifying and selectively propagating varieties rich in beta-carotene.

ii. Improving culinary procedure in order to avoid cooking losses and to ensure maximal retention.

iii. Develop more food based approaches and fortification.

iv. Popularising combinations of foods to be consumed for greater conversion.

v. Promoting acceptability and increased intake of green leafy vegetables and fruits through nutrition education.

References

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safety in humans. Int J Vit Nutr Res 1997; 67 : 71-90. 2. WHO - Global prevalence of vitamin-A deficiency, MDIS Working Paper No.2: Geneva : World Health Organisation, 1995: 1-11.

3. Vijayaraghavan K, Uma Nayak M. Availability and consumption pattern of carotene rich foods. In : Reddy V, Vijayaraghavan K., editors, Carotene rich foods for combating vitamin A deficiency. Hyderabad ::National Institute of Nutrition (ICMR), 1995: 10-6.

4. Vijayaraghavan K, Brahmam GNV, Gal Reddy Ch, Reddy V. Linking periodic dosing of vitamin A with universal immunization programme - An evaluation. Hyderabad National Institute of Nutrition (ICMR), 1996 : 35-8. 5. Food based approaches for solving micronutrient deficiencies. A manual for policy makers and programme planners. Rome : FAO & ILSI, 1995.

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37. Gronowska-Senger A, Wolf G. Effect of dietary protein on the enzyme from rat and human intestine which converts betacarotene to retinal. tI Nutr 1970; IDO : 300-8. 38. Pingle U, Sivakumar B. Effect of mango consumption on

vitamin A nutriture. Proc Nutr Soc India 1991; 37 : 373-9. 39. Vijayaraghavan K, Nayak MU, Bamji MS, Ramana GNV, Reddy Vinodini. Home gardening for combating vitamin A deficiency in rural India. Food Nutr Bull 1997;18 IS : 337-43.

B. Sivakumar

Division of Biophysics, National Institute of Nutrition (ICMR), Hyderabad

Reprint requests: Dr B. Sivakumar, Deputy Director (Sr Grade), Division of Biophysics, National Institute of Nutrition Jamai Osmania, Hyderabad 500007

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