Intestinal Scavenger Receptors Are Involved in Vitamin K1 Absorption

Vitamin K₁ (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K₁ was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K₁ absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K₁ uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K₁ uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K₁ intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.     

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

Menadione (Vitamin K3) Is a Catabolic Product of Oral Phylloquinone (Vitamin K1) in the Intestine and a Circulating Precursor of Tissue Menaquinone-4 (Vitamin K2) in Rats

Mice have the ability to convert dietary phylloquinone (vitamin K₁) into menaquinone-4 (vitamin K₂) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K₃), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and ¹H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.     

Ineffectiveness of phylloquinone epoxide as an inhibitor of prothrombin synthesis in the rat

Phylloquinone epoxide (vitamin K₁-oxide), a metabolite of phylloquinone, does not inhibit prothrombin synthesis when administered in high doses to Sprague-Dawley and warfarin-resistant rats. Further, it does not accumulate to presumed inhibitory levels in the livers of rats given physiological doses of ³H-phylloquinone when they are anticoagulated with warfarin. These data do not support the Bell-Matschiner hypothesis that warfarin exerts its action by inhibiting the vitamin K oxide reductase which results in the accumulation of vitamin K oxide and the inhibition of vitamin K at its active site. Rather, our data support the view that vitamin K and warfarin combine at different sites with a single regulatory protein which serves as a conformational switch for prothrombin synthesis.     

Prenylation and methylation reactions in phylloquinone (vitamin K1) synthesis in Capsicum annuum plastids

The biosynthesis of phylloquinone (vitamin K₁) was examined using Capsicum fruit chloroplasts and chromoplasts (apparently phylloquinone free). In both cases, the synthesis of phylloquinone from -naphthoquinone, dihydro--naphthoquinone, 1,4-dihydroxy-2-naphthoic acid (as precursors of the ring moiety) and (S)-adenosyl-L-methionine was achieved. In the presence of phytylpyrophosphate, the biosynthesis of phylloquinone in both organelles is particularly enhanced when 1,4-dihydroxy-2-naphthoic acid is used.     

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K₁ hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.     

Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection

A sensitive and precise method for measuring endogenous phylloquinone (K₁) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K₁ and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n=10) and cirrhosis (n=22) were significantly lower than in normal livers (n=6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.     

Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

Thematic Review Series: Fat-Soluble Vitamins: Vitamin K: Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.     

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K₁ 2,3-epoxide (K₁>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis–Menten constants of 1.20 μM for K₁>O and 260 μM for the active neutral form of THPP. The K_m value for K₁>O is in agreement with the value that we previously obtained using DTT (1.24 μM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K₁ and its previously reported base-catalyzed conversion to K₁, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO₂ and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.     

Vitamin K1 hydroquinone formation catalyzed by DT-diaphorase

Vitamin K₁ hydroquinone has been identified as a metabolite of vitamin K₁ biotransformation catalyzed by highly purified DT-diaphorase (NAD(P)H dehydrogenase, EC 1.6.99.2) isolated from livers of 3-methylcholanthrene induced rats. The hydroquinone was sufficiently stable to permit enzymatic reactions to be conducted under an atmosphere of air and quantitation of hydroquinone by high performance liquid chromatography. Based on kinetic data reported here, warfarin and probably dicoumarol at therapeutic levels do not appreciably affect DT-diaphorase catalyzed vitamin K hydroquinone formation.     

Detection and quantification of vitamin K(1) quinol in leaf tissues

Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone; vitamin K₁) is vital to plants. It is responsible for the one-electron transfer at the A₁ site of photosystem I, a process that involves turnover between the quinone and semi-quinone forms of phylloquinone. Using HPLC coupled with fluorometric detection to analyze Arabidopsis leaf extracts, we detected a third redox form of phylloquinone corresponding to its fully reduced - quinol-naphthoquinone ring (PhQH₂). A method was developed to quantify PhQH₂ and its corresponding oxidized quinone (PhQ) counterpart in a single HPLC run. PhQH₂ was found in leaves of all dicotyledonous and monocotyledonous species tested, but not in fruits or in tubers. Its level correlated with that of PhQ, and represented 5-10% of total leaf phylloquinone. Analysis of purified pea chloroplasts showed that these organelles accounted for the bulk of PhQH₂. The respective pool sizes of PhQH₂ and PhQ were remarkably stable throughout the development of Arabidopsis green leaves. On the other hand, in Arabidopsis and tomato senescing leaves, PhQH₂ was found to increase at the expense of PhQ, and represented 25-35% of the total pool of phylloquinone. Arabidopsis leaves exposed to light contained lower level of PhQH₂ than those kept in the dark. These data indicate that PhQH₂ does not originate from the photochemical reduction of PhQ, and point to a hitherto unsuspected function of phylloquinone in plants. The putative origin of PhQH₂ and its recycling into PhQ are discussed.     

Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay

Background

Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose–response data, usually summarized as half-maximal inhibitory concentration (IC₅₀), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data.

Methods

We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (K_m) and warfarin IC₅₀ for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC₅₀ to condition-independent K_i.

Results

Determination of the warfarin K_i specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay.

Conclusion

The K_i is not equal to the IC₅₀ value directly measured using the DTT-driven VKOR assay.

General significance

In contrast to warfarin IC₅₀ values determined in previous studies, warfarin inhibition expressed as K_i can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of K_i are required to accurately report and compare in vitro warfarin inhibition results.     

Vitamin K2 Biosynthetic Enzyme,UBIAD1 Is Essential for Embryonic Development of Mice

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K₂ biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 ^−/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 ^−/− embryonic stem (ES) cells failed to synthesize vitamin K₂ but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 ^+/− mice developed normally, exhibiting normal growth and fertility. Vitamin K₂ tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K₂ synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 ^+/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 ^−/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 ^+/− mice. These results suggest that UBIAD1 is responsible for vitamin K₂ synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K₂, but may have additional functions beyond the biosynthesis of vitamin K₂.     

Biosynthesis of vitamin B-12 Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K₁₂ and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[⁵⁸Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.     

The Pseudoenzyme PDX1.2 Boosts Vitamin B6 Biosynthesis under Heat and Oxidative Stress in Arabidopsis

Vitamin B₆ is an indispensable compound for survival, well known as a cofactor for numerous central metabolic enzymes and more recently for playing a role in several stress responses, particularly in association with oxidative stress. Regulatory aspects for the use of the vitamin in these roles are not known. Here we show that certain plants carry a pseudoenzyme (PDX1.2), which is involved in regulating vitamin B₆ biosynthesis de novo under stress conditions. Specifically, we demonstrate that Arabidopsis PDX1.2 enhances the activity of its catalytic paralogs by forming a heterododecameric complex. PDX1.2 is strongly induced by heat as well as singlet oxygen stress, concomitant with an enhancement of vitamin B₆ production. Analysis of pdx1.2 knockdown lines demonstrates that boosting vitamin B₆ content is dependent on PDX1.2, revealing that this pseudoenzyme acts as a positive regulator of vitamin B₆ biosynthesis during such stress conditions in plants.     

Are natural lipids UV-screening agents?

Summary Isolated lipids from Deinococcus radiodurans were reconstituted at final concentrations of 1 mg/ml into dioleoyl phosphatidyl choline (DOPC) vesicles and assayed for the ability to protect cells of Escherichia coli against killing by UV light (254 nm). Values of D₃₇ (UV dose required to reduce the number of surviving cells to 37% of the original number) were calculated from killing curves. E. coli was afforded the greatest protection with an individual lipid, identified as vitamin MK8 (D₃₇=310 J//m², compared to D₃₇=67 J/m² for E. coli irradiated in the presence of DOPC alone). Liposome-mediated protection was dependent on UV₂₅₄ absorbance and not on turbidity-related light-scattering. BOth vitamin MK8 from D. radiodurans and vitamin K₁, which is available commercially, showed a similar degree of UV₂₅₄-protection for E. coli. The UV-protective properties of vitamin K₁ were also investigated on mammalian cells in comparison with other natural lipids and known sunscreens. Survival curves were obtained for mouse fibroblast (L) cells irradiated at UV₂₅₄ in the absence or presence of DOPC liposomes into which were incorporated various natural lipids or standard sunscreen ingredients, all at final concentrations of 1 mg/ml. Experimentally determined values of D₃₇ were as follows: Vitamin K₁, 73 J/m²; \-carotene, 44 J/m₂; -tocopherol, 20 J/m₂; sulisobenzone, 156 J/m²; p-aminobenzoic acid (PABA), 113 J/m²; benzophenone, 80 J/m²; oxybenzone, 61 J/m² and DOPC alone. 23 J/m². Vitamin K₁, the most protective lipid tested, was also compared with PABA and oxybenzone (all at concentrations of 20 mg/ml; applied topicall) for its ability to protec Skh-hairless mice from UV₂₅₄-induced erythema, yielding a UV₂₅₄ protection factor of 3.5. In addition, vitamin K₁ (at 100 mg/ml) was able to provide hairless mice with a small degree of UVB protection, as indicated by an experimentally determined Solar Protection Factor of 1.5–2.0. Although it is concluded that vitamin K is not likely to account for the extraordinarily high degree of UV-resistance of D. radiodurans, vitamin K does show characteristics worthy of its consideration as a UV-screening agent. Offprint requests to: R. Anderson