Vitamin Basics

DSM Nutritional Products

Vitamin BasicsThe Facts about Vitamins in Nutrition

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Disclaimer

Copyright © by [DSM Nutritional Products AG] 2007

All rights reserved. No part of this publication may be reproduced, distri-buted or translated in any form or by any means, or stored in a database orretrieval system, without the prior written permission of the publisher. Themention of specific companies and trademarks does not imply that they areendorsed by DSM Nutritional Products AG (“DSM”). While making reaso-nable efforts to ensure that all information in this book is accurate and upto date, DSM makes no representation or warranty of the accuracy, reliabili-ty, or completeness of the information. The information provided herein is for informational purposes only. All medical information presented should be discussed with your healthcareprofessional. Remember, the failure to seek timely medical advice can haveserious ramifications. We strongly urge you to discuss any current healthrelated problems you are experiencing with a healthcare professional imme-diately. This publication does not constitute or provide scientific or medicaladvice, diagnosis, or treatment and is distributed without warranty of anykind, either express or implied. In no event shall DSM be liable for anydamages arising from the reader's reliance upon, or use of, these materials.The reader shall be solely responsible for any interpretation or use of thematerial contained herein.

Edited byDr. Volker Spitzer, Global Science Manager, DSM Nutritional Products Ltd.

With a foreword byProf. Dr. Florian Schweigert, President of the German Society for AppliedVitamin Research, Potsdam.

3rd edition 2007

(C) 1994, 1997, 2007 DSM Nutritional Products Ltd.

Designed by graphic art studio, Grenzach-Wyhlen, Germany

Printed in Germany Burger Druck, Waldkirch, Germany

2007 – REI – 50970 (1/0997.6.5)

DSM Nutritional Products is theworld’s largest supplier of nutritionalingredients, such as vitamins,carotenoids (antioxidants and pig-ments), other biochemicals and finechemicals, and premixes. The com-pany covers an unmatched breadthof applications in the area of ingre-dients, addressing the animal andhuman nutrition and health as wellas personal care industries.Starting in 1935 with the chemicalsynthesis of vitamin C, Roche grad-ually added other synthesized vita-mins to its range. In 2003, DSMacquired Roche's Vitamins and FineChemicals Division and today DSMNutritional Products sells the fullrange of fat-soluble and water-solu-ble vitamins, carotenoids, long chainpolyunsaturated fatty acids,enzymes, citric acid and nutraceuti-cals.

DSM OverviewHeadquartered in the Netherlands, DSM is active worldwide and develops,produces and sells innovative products and services that help improve thequality of life. DSM’s products are used in a wide range of end-markets andapplications, such as human and animal nutrition and health, personalcare, pharmaceuticals, automotive and transport, coatings, housing andelectrics & electronics. The group has annual sales of over 8 billion andemploys some 22,000 people. DSM ranks among the global leaders in manyof its fields.

Global operations:DSM Nutritional Products has 11 large production sites in 7 countries. Thecompany also runs 35 premix plants for Animal Nutrition and Health and 11premix plants for Human Nutrition and Health, where product combinationsare custom made to serve specific customer needs. DSM NutritionalProducts has some 40 sales offices that are active in over 100 countries. Itemploys approximately 6,200 people.

Research & Development: Building on its long tradition of industry leadership, DSM NutritionalProducts is committed to continuously providing outstanding products andservices for human and animal well-being. Most of these products arenature-identical, which means that their chemical structures and propertiescannot be distinguished from those found in plants or animals. R&D facili-ties are concentrated in the region of Basel, Switzerland, and are stronglyintegrated in an innovation network with other nutrition-related DSM R&Dcampuses in the Netherlands. Additionally, R&D satellites are managed inFrance and in China. In the area of process improvement, DSM makes everyeffort to keep the business’s main products competitive. The R&D strategyis based on the introduction of new chemical processes and the develop-ment of new biotechnology-based approaches. The latter efforts are sup-ported by advanced biotechnological techniques such as genomics andproteomics. DSM Nutritional Products supports its activities in vitamins andfine chemicals by conducting research that focuses primarily on processimprovement and the development of new products. Apart from developingnew products, DSM Nutritional Products is also working on improvedformulations and new combinations of existing products.

A pioneer in innovation:DSM Nutritional Products fosters innovation to the benefit of both the con-sumer’s future and that of the company. Lateral thinking and innovativeattitudes are valuable tools with which to secure that future. These lead todiscoveries that DSM then links to customers’ needs, extending the rangeof offering and creating new business opportunities.

DSM NUTRITIONAL PRODUCTS

Quality management:In 1991, DSM Nutritional Products introduced qualitymanagement based on Good Manufacturing Principles(GMP) and all the relevant International StandardsOrganization ISO (9000) quality standards. Since 1January 2002, the company has had a uniform andgroup-wide certification based on the new internationalstandard ISO 9001:2000. This means that all productionunits, premix plants, distribution centers and the entireglobal marketing organization are covered by the certifi-cate. All processes are designed to anticipate customerrequirements and market trends.

You can find more information on www.dsmnutritionalproducts.com

Products and servicesDSM Nutritional Products is the leading supplier ofvitamins, carotenoids and fine chemicals to the foodand pharmaceutical industries with a very strong globalmarketing and sales base. The company provides thefollowing products:

Carotenoidsb-CaroteneCaroCare® (b-Carotene – Natural Source)ApocarotenalApocarotenoic EsterCanthaxanthinLuteinredivivo™ (Lycopene)OPTISHARP™ (Zeaxanthin)

Fat soluble VitaminsVitamin A – Liquid and DryVitamin D3 – Liquid and DryVitamin E, Synthetic – Liquid and DryVitamin E, Natural SourceVitamin K1

Water soluble VitaminsVitamin B1 – ThiamineVitamin B2 – RiboflavinVitamin B3 – Niacin/NiacinamideVitamin B5 – PantothenatesPro-Vitamin B5 – PanthenolVitamin B6 – PyridoxineVitamin B12 – CyanocobalaminFolic AcidBiotinVitamin C

Long chain polyunsaturated fatty acidsROPUFA® (Omega-3 LC PUFA – Polyunsaturated FattyAcids)ROPUFA® (Omega-6 LC PUFA – Polyunsaturated FattyAcids)

NutraceuticalsALL-Q® (Coenzyme Q10)TEAVIGO™ (EGCG)BONISTEIN™ (Genistein)LAFTI® (Probiotics)HIDROX® (Olive Polyphenols)

Other ingredientsCitric AcidDextromethorphan Hydrobromide (DMH)Tretinoin

Micronutrient blends

Contents

Foreword 4

Introduction 5

Vitamin A 11

Beta-carotene 17

Vitamin D 23

Vitamin E 29

Vitamin K 35

Vitamin C 40

Vitamin B1 47

Vitamin B2 53

Vitamin B6 59

Vitamin B12 65

Niacin 71

Vitamin B5 76

Folic Acid 81

Biotin 87

References 93

Index 94

2

Foreword

While plants and micro organism have the capability toproduce the vitamins necessary for the metabolismthemselves, humans and animals have unfortunatelylost this ability during evolution. Because of the lack ofspecific enzymes for synthesis, vitamins became essen-tial nutrients for them. It was recognized more than3500 years ago that vitamins are essential food ingre-dients for maintaining health and well-being. The firstrecords related to the use of specific food items, as weknow today contain information on specific vitamins,such as vitamin A in liver, to prevent specific diseasessuch as night blindness. Only 3000 years later specificconditions of deficiency were recorded that could beattributed to the deficiency of selected nutrients. Wellknown examples are scurvy (vitamin C deficiency),beriberi (vitamin B1) and rickets (vitamin D). It tookanother 400 years until we ware able to attribute thesedisease conditions to specific active substances in ourdiet named then vitamins. Although we now know thatvitamins are not a uniform group of chemical sub-stances like proteins, carbohydrates and lipids, we stilluse the term to describe the whole group.

Since the beginning of the last century our knowledgeon the biological function of vitamins on the molecularand cellular level has increased significantly. Thisresearch is reflected by 20 Noble Prize winners between1928 and 1967. Despite intensive research efforts noadditional vitamins have been added to the list of 13vitamins accumulated between 1897 and 1941.

While in the past, scientist have basically been concern-ed with the role of vitamins in preventing vitamin relateddisease and their biochemical functions, today it isrecognized that vitamins have an important role inhealth and well-being beyond the mere prevention ofdeficiency. This aspect of vitamins is based on theobservation that vitamins are not only coenzymes inmetabolic processes but also act as potent antioxidantsand have hormone-like functions. The later is clearly

visible in the history of vitamin D research. In the late1970’s, research established vitamin D as a hormoneessential in bone metabolism. Based on such findings,vitamins are no longer classified into groups definedsimply by their physical-chemical properties – such aswater-soluble and fat-soluble vitamins. More appro-priately, vitamins are now classified according to theirbiological function in the body; vitamins with coenzymefunctions, vitamins with hormone-like properties andvitamins with antioxidants properties. But as expectedthe borderlines between these groups can not clearly bedefined and needs readjustment with the rapid progressin research.

In developing countries chronic, diet-related diseasesare still an important public health problem but in theaffluent societies, the prevention of degenerativediseases and also acute vitamin deficiencies might be ofconcern. Regarding the continuing debate of optimalvitamin levels and tolerable upper intake levels (UL) avalid knowledge-base of the daily expanding scientificevidence is necessary. This includes for example thedefinition of populations at risk, the problem of appro-priate biomarkers that not only reflect the dietary intakebut also the local status in specific tissues at risk ofdeficiency as well as environmental factors that influ-ence status and need for certain vitamins.

The following chapters of this book will contribute to thebetter understanding of the important role of vitaminsnot only in preventing specific deficiencies but alsomaintaining and improving human health and well-beingby summarizing the actual knowledge-base for the in-dividual vitamins.

Prof. Dr. Florian J. SchweigertPresident of the German Society for Applied VitaminResearch (GVF)Professor for Nutrition, University of PotsdamPotsdam, Germany

3

Introduction

Vitamins are essential organic nutrients required in very small amounts fornormal metabolism, growth and physical well-being. Most vitamins are notmade in the body, or only in insufficient amounts, and are mainly obtainedthrough food. When their intake is inadequate, vitamin deficiency disordersare the consequence. Vitamins are present in food in minute quantitiescompared to the macronutrients protein, carbohydrates and fat. The aver-age adult in industrialised countries eats about 600g of food per day on a -dry-weight basis, of which less than 1 gram consists of vitamins.

No single food contains all of the vitamins and, therefore, a balanced andvaried diet is necessary for an adequate intake. Each of the 13 vitaminsknown today has specific functions in the body, which makes every one ofthem unique and irreplaceable. Vitamins are essential for life!

Of the 13 vitamins, 4 are fat-soluble, namely vitamins A, D, E and K. Theother vitamins are water-soluble: vitamin C and the B-complex, consistingof vitamins B1, B2, B6, B12, folic acid, biotin, pantothenic acid and niacin.

The history of vitamins can be divided into five periods.

4

Vitamin Discovery Isolation Structure Synthesis

Vitamin A 1909 1931 1931 1947

Provitamin A (Beta-carotene) 1831 1930 1950

Vitamin D 1918 1932 1936 1959

Vitamin E 1922 1936 1938 1938

Vitamin K 1929 1939 1939 1939

Vitamin B1 1897 1926 1936 1936

Vitamin B2 1920 1933 1935 1935

Niacin 1936 1935 1937 1894

Vitamin B6 1934 1938 1938 1939

Vitamin B12 1926 1948 1956 1972

Folic Acid 1941 1941 1946 1946

Pantothenic Acid 1931 1938 1940 1940

Biotin 1931 1935 1942 1943

Vitamin C 1912 1928 1933 1933

Table 1: The History of Vitamins

1. The empirical healing of di-seases, now associated with vit-amin deficiency, through con-sumption of particular foods. Anexample is the use of liver to treatnight blindness (vitamin A defi-ciency) by the Egyptians (PapyrusEbers 1550-1570 BC), Assyrians,Chinese, Japanese, Greeks,Romans, Persians and Arabs.

2. The second phase was charac-terised by the ability to induce adeficiency disease in animals,which started with the classicalstudies of Lunin and Eijkmanaround 1890. The ability to pro-duce deficiency diseases, such asberiberi in animals, led toHopkins’ concept that smallamounts of “accessory growthfactors” are necessary for growthand life, and the coining of theterm “vitamine” in 1912 by thePolish-American scientist, Funk.

3. The third phase consisted inseven decades of excitingresearch involving the discovery,isolation, structure elucidationand synthesis of all the vitamins,and culminating in the synthesisof vitamin B12 in 1972. Most sci-entists think that the discovery of

any new vitamin is quite unlikely,although efforts are stil lcontinuing in that quest. Many ofthe researchers involved in thisgolden age of the vitaminsreceived a Nobel prize in reco-gnition of their great achie-vements (Table 2).

4. During the era of discovery, afourth period began which wasconcerned with the biochemicalfunctions, establishment ofdietary requirements andcommercial production. In theearly 1930s it was realised thatriboflavin (vitamin B2) was part ofthe “yellow enzyme”, which intime led to the elucidation of therole of the B-vitamins as coen-zymes. The subsequent identifi-cation of most of the B-vitaminsas coenzymes remained a centraltheme, defining their function formany decades. The first com-mercial synthesis of vitamin C byReichstein in 1933 was the startof a successful industrial effortthat led to the availabi l i ty ofrelatively inexpensive vitamins forresearch and use in animalfeedstuffs, for the fortification offood products, and for supple-ments.

5. The accumulation of reports of health benefits beyondpreventing deficiencies and excit-ing new biochemical functions ofvitamins ushered in a fifth period,starting with the report in 1955 ofthe cholesterol-lowering effect ofniacin (1). This is now a wellaccepted effect of the vitamin,which has nothing at all to do withits classical coenzyme role, and isa clear health effect beyond pre-venting the deficiency diseasepellagra.

Finally, work on the biochemicalfunction of vitamins in the last threedecades has considerably expandedour concept of how vitamins func-tion in the body and has helped pro-vide a chemical basis for the in vivoobservation of their health effects(Table 3).

Year Name Field Comments

1928 Adolf Windaus Chemistry for his research into the constitution of the steroids

and their connection with vitamins

1929 Christiaan Eijkman Medicine & for his discovery of the antineuritic vitamins

Physiology

Sir Frederick G. Hopkins Medicine & for his discovery of the growth stimulating vitamin

Physiology

1934 George R. Minot Medicine & for their discoveries concerning liver therapy of

William P. Murphy Physiology anaemias

George H. Whipple

1937 Sir Walter N. Haworth Chemistry for his research into the constitution of

carbohydrates and vitamin C

Paul Karrer Chemistry for his research into the constitution of carotenoids,

flavins and vitamins A and B2

Albert Szent-Györgyi Medicine & for his discoveries in connection with the biological

Physiology combustion processes, with particular reference to

vitamin C and the catalysis of fumaric acid

1938 Richard Kuhn Chemistry for his work on carotenoids and vitamins

1943 Carl Peter Henrik Dam Medicine & for his discovery of vitamin K

Physiology

Edward A. Doisy Medicine & for his discovery of the chemical nature of vitamin K

Physiology

1953 Fritz A. Lipmann Medicine & for his discovery of Coenzyme A and its importance

Physiology for intermediary metabolism

1955 Axel H.T. Theorell Medicine & for his discoveries concerning the nature and mode

Physiology of action of oxidation enzymes

1964 Konrad E. Bloch Medicine & for his discoveries concerning the mechanism and

Physiology regulation of cholesterol and fatty acid metabolism

Feodor Lynen Medicine & as above

Physiology

Dorothy C. Hodgkin Chemistry for her structural determination of vitamin B12

1967 Ragnar A. Granit Medicine & for his research, which illuminated the electrical

Physiology properties of vision by studying wavelength

discrimination in the eye

Halden K. Hartline Medicine & for his research on the mechanisms of sight

Physiology

George Wald Medicine & for his research on the chemical processes that

Physiology allow pigments in the retina of the eye to convert

light into vision

5

Table 2: Vitamin-Related Nobel Prize Winners

6

Vitamin Classical Role More Recent Role

Vitamin C Hydroxylation Reaction In Vivo Antioxidant

Beta-carotene Provitamin A Antioxidant, Immune Function

Vitamin K Clotting Factors Calcium Metabolism

Vitamin D Calcium Absorption, Differentiation and Growth,

Mineralisation of Bone Immune Function

Vitamin B6 Coenzyme Steroid Regulation

Niacin Coenzyme Lipid Lowering

Folic Acid Production and Maintenance Protection Against Neural Tube

of New Cells Birth Defects

Folic Acid, B6 and B12 Energy Metabolism May Lower Risk of Heart Disease and

Stroke*

Antioxidant vitamins Protection against Cancer and Heart

Disease*

Dietary ReferenceIntakes

From 1941 until 1989, RDAs(Recommended Dietary Allowances)were established and used to evalu-ate and plan menus to meet thenutrient requirements of certaingroups. They were also used inother applications such as interpre-ting food consumption records ofpopulations, establishing standardsfor food assistance programs,establishing guidelines for nutritionlabelling, etc.

The primary goal of RDAs was toprevent diseases caused by nutrientdeficiencies.

In the early 1990s, the Food andNutrition Board (FNB), the Instituteof Medicine, the National Academyof Sciences (USA), with the involve-ment of Health Canada, undertookthe task of revising the RDAs, and anew family of nutrient reference val-ues was born – the DietaryReference Intakes (DRIs).

The primary goal of having thesenew dietary reference values wasnot only to prevent nutrient deficien-cies, but also to reduce the risk ofchronic diseases such as osteo-porosis, cancer, and cardiovasculardisease.

The first report, Dietary ReferenceIntakes for Calcium, Phosphorus,Magnesium, Vitamin D and Fluoride,was published in 1997. Since then,three additional vitamin relatedreports have been released,addressing folate and other B vit-amins, dietary antioxidants (vitaminsC, E, selenium and the carotenoids),and the micronutrients (vitamins A,K, and trace elements such as iron,iodine, etc). The DRIs are a compre-hensive scientific source primarily fornutrition scientists (see References).They are used by health authoritiesin many countries as a basis fordecisions regarding nutritional infor-mation on micronutrients.There are four types of DRI referencevalues: the Estimated AverageRequirement (EAR), the Recom-mended Dietary Allowance (RDA), theAdequate Intake (AI) and the TolerableUpper Intake Level (UL).

• Estimated Average Requirement(EAR) – the amount of a nutrientthat is estimated to meet therequirement of half of all healthyindividuals in a given age andgender group. This value is basedon a thorough review of the scien-tific literature.

• Recommended Dietary Allowance(RDA) – the average daily dietaryintake of a nutrient that is sufficientto meet the requirement of nearlyall (97-98%) healthy persons. Thisis the number to be used as a goalfor individuals. It is calculated fromthe EAR.

• Adequate Intake (AI) – only estab-lished when an EAR (and thus anRDA) cannot be determinedbecause the data are not clear-cutenough; a nutrient has either anRDA or an AI. The AI is based onexperimental data or determinedby estimating the amount of anutrient eaten by a group ofhealthy people and assuming thatthe amount they consume is ade-quate to promote health.

Table 3: Biochemical Function of Vitamins

*Research ongoing

7

• Tolerable Upper Intake Level (UL)– the highest continuing dailyintake of a nutrient that is likely topose no risks of adverse healtheffects for almost all individuals. Asintake increases above the UL, therisk of adverse effects increases.Consistently consuming a nutrientat the upper level should not causeadverse effects. Intake levels at theUL can be interpreted as a ‘war-ning flag’, not as reason for alarm.

Certain groups atrisk of vitamindeficienciesWith the advent of vitamin fortifica-tion in the manufacturing of flour,cereals and other foods, specificvitamin deficiency diseases such asscurvy, beriberi, rickets and pellagrahave become rare in most industri-alised countries. However, in manyAfrican, Asian and Latin Americancountries, chronic, diet-related di-seases continue to be a major healthproblem. In these countries there isa need to eliminate frank vitamin A,C and B-complex deficiencies, aswell as other micronutrient deficien-cies (iodine, iron, selenium, zinc andcalcium).

However, even in highly industri-alised countries, numerous largegovernment nutrition surveys of thepopulation indicate that marginalvitamin deficiencies with unspecificsymptoms, like fatigue and frequentheadaches, are probably not rare.They are difficult for the individual todetect and are largely ignored.Marginal vitamin deficiency is a“state of gradual vitamin depletion inwhich there is evidence of personallack of well-being associated withimpairment of certain biochemicalreactions”. Studies have found thatmany people have nutritional defi-ciencies which do not show up in aroutine physical examination. It has

also been suggested that marginaldeficiencies are linked to behaviour-al and physiological changes.Extensive surveys have revealed thatmore than 60% of the elderly havedeficient dietary intake of vitamin D,E and folate. Other vitamins – criticalnot just for the elderly – includethiamin (B1), panthothenic acid, andbiotin.

Many individuals have healthproblems, habits, or living situationsin which chronic or periodic intake of vitamins should exceed theordinary requirement. High-risk-groups include:

• the elderly• adolescents• young or pregnant and lactating

women• alcoholics• cigarette smokers• vegetarians• people fasting or on dietary

intervention• laxative abusers• users of contraceptives and

analgesics and other medicationsfor chronic disease

• people with specific disorders ofthe gastrointestinal tract.

However, marginal deficiencies arenot only limited to those groupslisted. The gradual change in theway we live has influenced our dietsand has altered our habitual intakeof vitamins and minerals. Hecticlifestyles, reduced physical activityand an increase in fast and conve-nience food have all played a signifi-cant role. As a result, a significantproportion of the population fails toreach recommended intake levels.

AntioxidantVitamins

Vitamin C, vitamin E and caro-tenoids, such as beta-carotene, aremicronutrients with antioxidantproperties. Antioxidants are sub-stances that prevent oxidation orchemical reactions involving oxygen.

As the atmosphere changed frombeing anaerobic to aerobic, oxygenbecame available in energy pro-duction for living organisms, but italso carried a price. When energy isproduced, unstable oxygen speciesknown as free radicals are formed.Free radicals are also produced atother sites in the metabolism (e.g.,by activated phagocytes as part ofthe immune defence), and throughexogenous sources such as expo-sure to cigarette smoke, environ-mental pollutants and ultravioletlight. Free radicals are atoms or mo-lecules that have an unpaired elec-tron which makes them very reac-tive. They have the potential to dam-age DNA, proteins, carbohydrates,lipids and cell membranes. In addi-tion to free radicals, there is anotherhighly reactive compound that is apotent generator of free radicals: it iscalled singlet oxygen. This moleculeis unique in that it contains a pair ofelectrons but exists in an unstableconfiguration and is very reactive.

The body has an elaborate antioxi-dant defence system that works toneutralise free radicals and otherhighly reactive species. The majorbiological antioxidants are enzymes(superoxide dismutase, catalase andglutathione peroxidase) as well asnon-enzymatic scavengers (such asuric acid, CoQ10, glutathione, thiolsin proteins) and the antioxidantvitamins (beta-carotene, vitamin Cand E).

Each of the antioxidant nutrients hasspecific characteristics, and theyoften work synergistically to

8

strengthen the overall antioxidantcapability of the body.

Vitamin E is the principal fat-solubleantioxidant in the body and isresponsible for protecting thepolyunsaturated fatty acids in cellmembranes from oxidation by freeradicals. Vitamin E exhibits a sparingeffect on beta-carotene by pro-tecting the conjugated double bondsfrom being oxidised. Exposure toincreased oxygen levels, such asreperfusion, results in free radical-mediated tissue damage. However,due to the capability of vitamin E towork at higher oxygen pressures,free radicals are scavenged andtissue injury is minimised.

Beta-carotene also has antioxidantproperties and is one of the mostpowerful quenchers of singlet oxy-gen. It can dissipate the energy ofsinglet oxygen, thus preventing thisactive molecule from generating freeradicals.

Vitamin C, a water-soluble antioxi-dant, interacts with free radicals inthe aqueous compartment of cells.Additionally, vitamin C is consideredthe most important antioxidant inextra-cellular fluids. Vitamin C hasthe ability to regenerate vitamin Eafter it has neutralised free radicalsand terminated chain reactions.

The balance of free radical produc-tion and the level of antioxidantdefences have important diseaseand health implications. If there aretoo many free radicals produced,and too few antioxidants, to a condi-tion of “oxidative stress” developswhich can lead to chronic injury.

It has therefore been suggested thatoxidative stress might play a role inthe development of a number ofdiseases:

• cancer • atherosclerosis• cardiovascular diseases• cataracts• age-related macular degeneration• Alzheimer’s disease• immune dysfunction• rheumatoid arthritis

Oxidative stress also plays a rolein the aging process.

The scientific literature containsmany research articles on thepotential roles of the antioxidant

nutrients in disease prevention.Many studies are just beginning

while others continue to show thepositive effects of the antioxidant

nutrients. It therefore seemsprudent to ensure an ade-

quate intake of beta-carotene, vitamin C

and vitamin E in thediet or throughsupplementation.

Vitamins continueto fascinate, and

have become thefocus of renewed

attention on the part of researchers,health/nutrition professionals, andgovernment policymakers, as well asthe general public.

References

http://riley.nal.usda.gov/nal_display/index.php?inf

o_center=4&tax_level=3&tax_subject=256&topic_

id=1342&level3_id=5141

Dietary Reference Intakes for Calcium, Phos-

phorus, Magnesium, Vitamin D, and Fluoride

(1997) National Academy of Sciences. Institute of

Medicine. Food and Nutrition Board.

Dietary Reference Intakes for Thiamin, Riboflavin,

Niacin, Vitamin B6, Folate, Vitamin B12,

Pantothenic Acid, Biotin, and Choline (1998)

National Academy of Sciences. Institute of

Medicine. Food and Nutrition Board.

Dietary Reference Intakes for Vitamin C, Vitamin E,

Selenium, and Carotenoids (2000) National

Academy of Sciences. Institute of Medicine. Food

and Nutrition Board.

Dietary Reference Intakes for Vitamin A, Vitamin K,

Arsenic, Boron, Chromium, Copper, Iodine, Iron,

Manganese, Molybdenum, Nickel, Silicon,

Vanadium, and Zinc (2001) National Academy of

Sciences. Institute of Medicine. Food and Nutrition

Board.

9

Vitamin A

SynonymsRetinol, axerophthol

ChemistryRetinol and its related compounds consist of four isoprenoid units joinedhead to tail and contain five conjugated double bonds. They naturally occuras alcohol (retinol), as aldehyde (retinal) or as acid (retinoic acid).

CH3 CH3

CH2OH

CH3

CH3CH3

Molecular formula of vitamin A (retinol)

Vitamin A crystals in polarised light

10

Introduction

Vitamin A is a generic term for agroup of lipid soluble compoundsrelated to retinol. Retinol is oftenreferred to as preformed vitamin A. Itis found only in animal sources,mainly as retinyl esters and in foodsupplements. Many cultures haveused ox liver as an excellent sourceof vitamin A to cure night blindness.The liver was first pressed to the eyeand then eaten; the Egyptiansdescribed this cure at least 3,500years ago. Beta-carotene and othercarotenoids that can be convertedto vitamin A by an enzymaticprocess in the body are referred toas provitamin A. They are found onlyin plant sources.

Functions

Retinal, the oxidised metabolite ofretinol, is required for the process ofvision. Retinoic acid, another vitaminA metabolite, is considered to beresponsible for all non-visual func-tions of vitamin A. Retinoic acidcombines with specific nuclearreceptor proteins which bind to DNAand regulate the expression of vari-ous genes, thereby influencingnumerous physiological processes.Retinoic acid is therefore classifiedas a hormone.

VisionReceptor cells in the retina of theeye (rod cells) contain a light-sensi-tive pigment called rhodopsin, whichis a complex of the protein opsinand the vitamin A metabolite retinal.The light-induced disintegration ofthe pigment triggers a cascade ofevents which generate an electricalsignal to the optic nerve. Rhodopsincan only be regenerated from opsinand vitamin A. Rod cells with thispigment can detect very smallamounts of l ight, making themimportant for night vision.

Cellular differentiationThe many different types of cells inthe body perform highly specialisedfunctions. The process wherebycells and tissues become “pro-grammed” to carry out their specialfunctions is called differentiation.Through the regulation of geneexpression, retinoic acid plays amajor role in cellular differentiation.Vitamin A is necessary for normaldifferentiation of epithelial cells, thecells of all tissues lining the body,such as skin, mucous membranes,blood vessel walls and the cornea.In vitamin A deficiency, cells losetheir ability to differentiate properly.

Growth and developmentRetinoic acid plays an important rolein reproduction and embryonicdevelopment, particularly in thedevelopment of the spinal cord andvertebrae, limbs, heart, eyes andears.

Immune functionVitamin A is required for the normalfunctioning of the immune systemand therefore helps to protectagainst infections in a number ofways. It is essential in maintaining

Food Vitamin A (Retinol) RE

µg/100g µg/100g

Veal liver 28000 28000

Carrots - 1500

Spinach - 795

Melon (cantaloupe) - 784

Butter 590 653

Cheese (Cheddar) 390 440

Egg 276 272

Broccoli - 146

Salmon 41 41

Milk (whole) 35 35

Vitamin A content of foods

(Souci, Fachmann, Kraut)

11

the integrity and function of the skinand mucosal cells, which function asa mechanical barrier and defend thebody against infection. Vitamin Aalso plays a central role in the devel-opment and differentiation of whiteblood cells, such as lymphocytes,killer cells and phagocytes, whichplay a critical role in the defence ofthe body against pathogens.

Main functions in a nutshell:• Vision• Reproduction• Growth and development• Cellular differentiation• Immune function

Dietary sources

The richest food source of prefor-med vitamin A is l iver, with considerable amounts also found inegg yolk, whole milk, butter andcheese. Provitamin A carotenoidsare found in carrots, yellow and darkgreen leafy vegetables (e.g. spinach,broccoli), pumpkin, apricots andmelon.Until recently, vitamin A activity infoods was expressed as interna-tional units (IU). This is still themeasurement generally used onfood and supplement labels. In orderto standardise vitamin A measure-ment, it has now been international-ly agreed to state vitamin A activityin terms of a new unit called theretinol equivalent, or RE, whichaccounts for the rate of conversionof carotenoids to retinol.

1 RE = 1 µg retinol= 6 µg beta-carotene= 12 µg other provitamin A

carotenoids= 3.33 IU vitamin A activity

from retinol

Absorption andbody stores

Vitamin A is absorbed in the upperpart of the small intestine. Pro-vitamin A carotenoids can becleaved into retinol via an enzymaticprocess. Preformed vitamin Aoccurs as retinyl esters of fattyacids. They are hydrolysed andretinol is absorbed into intestinalmucosal cells (i.e. enterocytes). Afterre-esterification it is incorporatedinto chylomicrons, excreted intolymphatic channels, delivered to theblood and transported to the liver.Vitamin A is stored in the liver asretinyl esters; stores are enough forone to two years in most adultsliving in industrialised countries.

Measurement

Vitamin A can be measured in theblood and other body tissues by various modern techniques. Forrapid field tests, a method has beendeveloped recently using driedblood spots. Typical serum level is1.1-2.3 µmol/L. According to theWHO, plasma levels of #0,35 µmol/Lindicate a vitamin A deficiency.

Stability

Vitamin A is sensitive to oxidation by air. Loss of activity is accelerated by heat and exposure to light.Oxidation of fats and oils (e.g. butter, margarine, cooking oils) candestroy fat soluble vitamins includ-ing vitamin A. The presence ofantioxidants such as vitamin E there-fore contributes to the protection ofvitamin A.

InteractionsPositive interactions• Vitamin E protects vitamin A from

being oxidised; hence, adequatevitamin E status protects vitamin Astatus.

Negative interactions• Disease and infection, especially

measles, compromise vitamin Astatus and conversely, poorvitamin A status decreasesresistance to diseases.

• Chronic heavy alcohol intake canimpair liver storage of vitamin A.

• Acute protein deficiency interfereswith vitamin A metabolism; simi-larly, too little fat in the diet inter-feres with the absorption of bothvitamin A and carotenoids.

• Vitamin A deficiency may result inimpaired iron absorption anddecrease its utilisation for erythro-poiesis, thereby potentially exacer-bating iron deficiency anaemia.

• Zinc deficiency may adverselyaffect mobilisation of vitamin Afrom hepatic stores and absorp-tion of vitamin A from the gut.

12

DeficiencyVitamin A deficiency is rare in theWestern world, but in developingcountries it is one of the most wide-spread, yet preventable, causes ofblindness. The earliest symptom ofvitamin A deficiency is impaired darkadaptation, or night blindness.Severe deficiency causes a condi-tion called xerophthalmia, charact-erised by changes in the cells of thecornea that ultimately result incorneal ulcers, scarring and blind-ness. The appearance of skinlesions (follicular hyperkeratosis) isalso an early indicator of inadequatevitamin A status. Growth retardationis a common sign in children.Because vitamin A is required for thenormal functioning of the immunesystem, even children who are onlymildly deficient in vitamin A have ahigher incidence of respiratory dis-ease and diarrhoea, as well as ahigher rate of mortality from infec-tious diseases, than children whoconsume sufficient vitamin A.Some diseases may themselvesinduce vitamin A deficiency, mostnotably liver and gastrointestinal dis-eases, which interfere with theabsorption and utilisation of vitamin A. Vitamin A deficiency during pregnancyleads to malformations during foetaldevelopment.

Disease preventionand therapeuticuse

Studies have shown that vitamin Asupplementation given to childrenaged over 6 months reduces all-cause mortality by between 23%and 30% in low income countries. Thebeneficial effect is assumed to be dueto the prevention of vitamin A deficien-cy. The World Health Organisation(WHO) recommends that supplementsshould be given when children arevaccinated. The currently recom-mended doses are 100,000 IU at age6-11 months and 200,000 IU at age $ 12 months every 3-6 months. Xerophthalmia is treated with highdoses of vitamin A (50,000-200,000IU according to age).In developing countries, wherevitamin A deficiency is one of themost serious health problems,children under the age of 6 yearsand pregnant and lactating womenare the main vulnerable groups.Since vitamin A can be stored in theliver, it is possible to build up a

reserve in children by administrationof high-potency doses. In regularperiodic distribution programmes forthe prevention of vitamin Adeficiency, infants < 6 months of agereceive a dose of 50,000 IU ofvitamin A, and children between sixmonths and one year receive100,000 IU every 4-6 months, whilechildren > 12 months of age receive200,000 IU every 4-6 months. Asingle dose of 200,000 IU given tomothers immediately after delivery oftheir child has been found to in-crease the vitamin A content ofbreast milk. However, caution isnecessary when considering vitaminA therapy for lactating women, oth-erwise a co-existing pregnancy maybe endangered: during pregnancy, adaily dose of 10,000 IU vitamin Ashould not be exceeded.Administration of high doses ofvitamin A to children with measlescomplications, but no overt signs ofvitamin A deficiency, decreasesmortality by over 50% and signifi-cantly lowers morbidity.Natural and synthetic vitamin A ana-logues have been used to treat pso-riasis and severe acne.

Child suffering from corneal scar

Current recommendations in the USA

RDA*

Infants # 6 months 400 µg (Adequate Intake, AI)

Infants 7–12 months 500 µg (AI)

Children 1–3 years 300 µg



Males $ 14 years 900 µg

Females $ 14 years 700 µg

Pregnancy 14-18 years 750 µg

Pregnancy $ 19 years 770 µg

Lactation 14–18 years 1,200 µg

Lactation $ 19 years 1,300 µg

*The Dietary Reference Intakes (DRIs) are actually

a set of four reference values: Estimated Average

Requirements (EAR), Recommended Dietary

Allowances (RDA), Adequate Intakes (AI), and

Tolerable Upper Intake Levels, (UL) that have

replaced the 1989 Recommended Dietary

Allowances (RDAs). The RDA was established as

a nutritional norm for planning and assessing

dietary intake, and represents intake levels of

essential nutrients considered to meet adequately

the known needs of practically all healthy people

13

RecommendedDietary Allowance(RDA)

The recommended daily intake ofvitamin A varies according to age,sex, risk group and other criteriaapplied in individual countries (700–1000 µg RE/day for men, 600–800 µg RE/day for women. Inthe USA the RDA for adults is 900µg (men) and 700 µg (women) perday of preformed vitamin A (retinol).During lactation, an additional 500–600 µg per day are recom-mended. Infants and children, due totheir smaller body size, have a lowerRDA than adults.

Safety

Because vitamin A (as retinyl ester) isstored in the liver, large amounts takenover a period of time can eventuallyexceed the liver's storage capacity,spill into the blood, and produceadverse effects (liver damage, boneabnormalities and joint pain, alopecia,headaches, vomiting, and skindesquamation). Hypervitaminosis Acan occur acutely following very highdoses taken over a period of severaldays, or as a chronic condition fromhigh doses taken over a long period oftime. Thus, there is concern about thesafety of high intakes of preformedvitamin A (retinol), especially forinfants, small children, and women ofchildbearing age.Normal foetal development requiressufficient vitamin A intake, butconsumption of excess retinol duringpregnancy is known to cause malfor-mations in the newborn.Several recent prospective studiessuggest that long-term intakes of pre-formed vitamin A in excess of 1,500µg/day are associated with increasedrisk of osteoporotic fracture anddecreased bone mineral density inolder men and women. Only excess

intakes of preformed vitamin A, notbeta-carotene, were associated withadverse effects on bone health.Current levels of vitamin A in fortifiedfoods are based on RDA levels, ensur-ing that there is no realistic possibilityof vitamin A overdosage in the gener-al population. In the vast majority ofcases, signs and symptoms of toxicityare reversible upon cessation of vita-min A intake. Beta-carotene is consid-ered a safe form of vitamin A becauseit is converted by the body only asneeded.

The Food and Nutrition Board of theInstitute of Medicine (2001) and theEC Scientific Committee on Food(2002) have set the tolerable upperintake level (UL) of vitamin A intake foradults at 3000 µg RE/day with appro-priately lower levels for children.

Supplements andfood fortification

Vitamin A is available in soft gelatinecapsules, as chewable or efferves-cent tablets, or in ampoules. It isalso included in most multivitamins.Retinyl acetate, retinyl palmitate andretinal are the forms of vitamin Amost commonly used in supple-ments.Margarine and milk are commonlyfortif ied with vitamin A. Beta-carotene is added to margarine andmany other foods (e.g. fruit drinks,salad dressings, cake mixes, icecream) both for its vitamin A activityand as a natural food colourant.

Industrial production

Nowadays vitamin A is rarelyextracted from fish liver oil. Themodern method of industrial synthe-sis of nature-identical vitamin A is ahighly complex, multi-step process.

14

History

Although it has been known since ancient Egyptian times that certain foods,such as liver, would cure night blindness, vitamin A per se was not identi-fied until 1913. Its chemical structure was defined by Paul Karrer in 1931.Professor Karrer received a Nobel Prize for his work because this was thefirst time that a vitamin’s structure had been determined.

1831 Wackenroder isolates the orange-yellow colourant from carrotsand names it “carotene.”

1876 Snell successfully demonstrates that night blindness and xeroph-thalmia can be cured by giving the patient cod liver oil.

1880 Lunin discovers that, besides needing carbohydrates, fats andproteins, experimental animals can only survive if given smallquantities of milk powder.

1887 Arnaud describes the widespread presence of carotenes inplants.

1909 Stepp successfully extracts the vital liposoluble substance frommilk.

1915 McCollum differentiates between “fat-soluble A” and “water-soluble B.”

1929 The vitamin A activity of beta-carotene is demonstrated in animalexperiments.

1931 Karrer isolates practically pure retinol from the liver oil of aspecies of mackerel. Karrer and Kuhn isolate active carotenoids.

1946 Isler undertakes the first large-scale industrial synthesis of vita-min A.

1984 Sommer demonstrates that vitamin A deficiency is a major causeof infant mortality in Indonesia.

1987 Chombon in Strasbourg and Evans in San Diego, and theirrespective coworkers, simultaneously discover the retinoic acidreceptors in cell nuclei.

1997 UNICEF, the World Health Organisation (WHO), and the govern-ments of countries including Canada, the United States and theUnited Kingdom, as well as national governments in countrieswhere vitamin deficiency is widespread, launch a global cam-paign to distribute high-dose vitamin A capsules to malnourishedchildren.

Paul Karrer

Otto Isler

Elmer V. McCollum

15

Beta-carotene

ChemistryBeta-carotene is a terpene. It is made up of eight isoprene units, which arecyclised at each end. The long chain of conjugated double bonds is respon-sible for the orange colour of beta-carotene.

CH3 CH3

CH3

CH3H3C

CH3 CH3CH3 CH3

H3C

Molecular formula of beta-carotene

Beta-carotene crystals in polarised light

16

IntroductionBeta-carotene is one of more than600 carotenoids known to exist innature. Carotenoids are yellow,orange and red pigments that arewidely distributed in plants. In 1831,beta-carotene was isolated byWackenroder. Its structure was deter-mined by Karrer in 1931, whoreceived a Nobel prize for his work.About 50 of the naturally occurringcarotenoids can potentially yield vita-min A and are thus referred to asprovitamin A carotenoids. Beta-carotene is the most abundant andmost efficient provitamin A in ourfoods.Currently available evidence suggeststhat in addition to being a source ofvitamin A, beta-carotene plays manyimportant biological roles that may beindependent of its provitamin status.

Functions

Beta-carotene is the main safedietary source of vitamin A. Vitamin Ais essential for normal growth anddevelopment, immune system func-tion, and vision.Beta-carotene can quench singletoxygen, a reactive molecule that isgenerated, for instance, in the skin byexposure to ultraviolet light, andwhich can induce precancerouschanges in cells. Singlet oxygen iscapable of triggering free radicalchain reactions.

Beta-carotene has antioxidant prop-erties that help neutralise free radi-cals – reactive and highly energisedmolecules which are formed throughcertain normal biochemical reactions(e.g. the immune response,prostaglandin synthesis), or throughexogenous sources such as air pollu-tion or cigarette smoke. Free radicalscan damage lipids in cell membranesas well as the genetic material incells, and the resulting damage maylead to the development of cancer.

Main functions in a nutshell:• Provitamin A• Antioxidant activity

Dietary sources

The best sources of beta-caroteneare yellow/orange vegetables andfruits and dark green leafy vegeta-bles:

• Yellow/orange vegetables – car-rots, sweet potatoes, pumpkins,winter squash

• Yellow/orange fruits – apricots,cantaloupes, papayas, mangoes,carambolas, nectarines, peaches

• Dark green leafy vegetables –spinach, broccoli, endive, kale,chicory, escarole, watercress andbeet leaves, turnips, mustard,dandelion

• Other good vegetable and fruitsources – summer squash,asparagus, peas, sour cherries,prune plums.

The beta-carotene content of fruitsand vegetables can vary accordingto the season and degree of ripen-ing.


Bile salts and fat are needed for theabsorption of beta-carotene in theupper small intestine. Many dietaryfactors, e.g. fat and protein, affectabsorption. Approximately 10-50%of the total beta-carotene consumedis absorbed in the gastrointestinaltract. The proportion of carotenoidsabsorbed decreases as dietaryintake increases. Within the intestin-al wall (mucosa), beta-carotene ispartially converted into vitamin A(retinol) by the enzyme dioxygenase.This mechanism is regulated by theindividual's vitamin A status. If thebody has enough vitamin A, the con-version of beta-carotene decreases.Therefore, beta-carotene is a verysafe source of vitamin A and highintakes will not lead to hypervita-minosis A.Excess beta-carotene is stored inthe fat tissues of the body and theliver. The adult's fat stores are oftenyellow from accumulated carotenewhile the infant's fat stores arewhite.

Bioavailability of beta-caroteneBioavailability refers to the pro-portion of beta-carotene that canbe absorbed, transported andutilised by the body once it hasbeen consumed. It is influencedby a number of factors:• Beta-carotene from dietary

supplements is better absorbedthan beta-carotene from foods

• Food processing such aschopping, mechanical homo-genisation and cooking en-hances bioavailability of beta-carotene

• The presence of fat in the intes-tine affects absorption of beta-carotene. The amount of dietaryfat required to ensure caro-tenoid absorption seems to below (approximately 3-5g permeal)

Food Beta-carotene (mg/100g)

Carrots 7.6

Kale 5.2

Spinach 4.8

Cantaloupes 4.7

Apricots 1.6

Mangoes 1.2

Broccoli 0.9

Pumpkins 0.6

Asparagus 0.5

Peaches 0.1

Beta-carotene content of foods


17

MeasurementPlasma carotenoid concentration isdetermined by HPLC. It reflects theintake of carotenoids. Traditionally,vitamin A activity of beta-carotenehas been expressed in InternationalUnits (IU; 1 IU = 0.60 µg of all-transbeta-carotene). However, this con-version factor does not take intoaccount the poor bioavailability ofcarotenoids in humans. Thus, theFAO/WHO Expert Committee pro-posed that vitamin A activity beexpressed as retinol equivalents(RE). 6 µg beta-carotene provide 1µg retinol.For labelling, official national direc-tives should be followed.

1 RE = 1 µg retinol = 6 µg beta-carotene= 3.33 IU vitamin A activity

from retinol= 10 IU vitamin A activity

from beta-carotene

StabilityCarotenoids can lose some of theiractivity in foods during storage dueto the action of enzymes and expo-sure to light and oxygen. Dehy-dration of vegetables and fruits maygreatly reduce the biological activityof carotenoids. On the other hand,carotenoid stability is retained infrozen foods.

Interactions

Negative interactionsCholestyramine and colestipol (cho-lesterol-lowering agents), mineral oil,orlistat (a weight loss medication)and omeprazole (proton-pumpinhibitor) can reduce absorption ofcarotenoids.

Deficiency

Although consumption of provitaminA carotenoids can prevent vitamin Adeficiency, there are no knownadverse clinical effects of a lowcarotenoid diet, provided vitamin Aintake is adequate.

Disease prevention andtherapeutic use

Immune systemIn a number of animal and humanstudies beta-carotene supplementa-tion was found to enhance certainimmune responses. Early studiesdemonstrated the ability of beta-carotene and other carotenoids toprevent infections. Some clinical tri-als have found that beta-carotenesupplementation improves severalbiomarkers of immune function. Itcan lead to an increase in the num-ber of white blood cells and theactivity of natural killer cells. Both ofthese are important in combatingvarious diseases. It may be the casethat beta-carotene stimulates theimmune system once it has under-gone conversion to vitamin A.Another explanation could be thatthe antioxidant actions of beta-carotene protect cells of the immunesystem from damage by reducingthe toxic effects of reactive oxygenspecies.

SkinRecent evidence points to a role ofbeta-carotene in protecting the skinfrom sun damage. Beta-carotenecan be used as an oral sun protec-tant in combination with sunscreensfor the prevention of sunburn. Itseffectiveness has been proven bothalone and in combination with othercarotenoids or antioxidant vitamins.

Cancer and cardiovascular dis-easesEpidemiological studies consistentlyindicate that as consumption ofbeta-carotene-rich fruits and veg-etables increases, the risk of certaincancers (i.e. lung and stomach can-cer) and cardiovascular diseasesdecreases.Additionally, animal experimentshave shown that beta-carotene actsas a cancer risk reduction agent.

18

This is further supported by studiesof biomarkers for the developmentof certain cancers. There is no evi-dence that beta-carotene supple-mentation reduces the risk of cardio-vascular diseases.

Erythropoietic protoporphyriaIn patients with erythropoietic proto-porphyria – a photosensitivity disor-der leading to abnormal skin reac-tions to sunlight – beta-carotene indoses of up to 180 mg has beenshown to exert a photoprotectiveeffect.


Until now, dietary intake of beta-carotene has been expressed aspart of the RDA for vitamin A. Thedaily vitamin A requirements foradult men and women are 900 µgand 700 µg of preformed vitamin A(retinol) respectively (FNB, 2001).Apart from its provitamin A function,data continue to accumulate sup-porting a role for beta-carotene asan important micronutrient in its ownright. Consumption of foods rich inbeta-carotene is being recommend-ed by scientific and governmentorganisations such as the USNational Cancer Institute (NCI) andthe US Department of Agriculture(USDA). If these dietary guidelinesare followed, dietary intake of beta-carotene (about 6 mg) would be sev-eral times the average amountpresently consumed in the US(about 1.5 mg daily).

Safety

Beta-carotene is a safe source ofvitamin A. Due to the regulated con-version of beta-carotene into vitamin

A, overconsumptiondoes not pro-duce hypervita-minosis A.Excessive intakesof beta-carotene maycause carotenodermia,which manifests itself ina yellowish tint of theskin, mainly in the palms of thehands and soles of the feet. The yel-low colour disappears whencarotenoid consumption is reducedor stopped. High doses of beta-carotene (up to180 mg/day) used for the treatmentof erythropoietic protoporphyriahave shown no adverse effects.In two studies investigating theeffect of beta-carotene supplemen-tation on the risk of developing lungcancer, an apparent increase of lungcancer in chronic heavy smokerswith intakes of more than 20 mg/dayover several years has beenobserved.The reasons for these find-ings are not yet clear.The British Expert Committee onVitamins and Minerals (EVM) recom-mends a Safe Upper Level for sup-plementation of 7 mg/day over a life-time period. Other agencies such asthe European DACH Society(German Society of Nutrition,Austrian Society of Nutrition, SwissSociety of Nutrition Research) haveconcluded that a daily intake of upto 10 mg of beta-carotene is safe.


Beta-carotene is available in hardand soft gelatine capsules, in multi-vitamin tablets, and in antioxidantvitamin formulas and as food colour.Margarine and fruit drinks are oftenfortified with beta-carotene. In 1941,the US Food and Drug Admini-stration (FDA) established a stand-ard of identity for the addition ofvitamin A to margarine; since

then, however, vitamin A has beenpartly replaced by beta-carotene, which additionally imparts an attrac-tive yellowish colour to this product.Due to its high safety margin, beta-carotene has been recognised asmore suitable for fortification pur-poses than vitamin A.


Isler and coworkers developed amethod to synthesise beta-carotene, and it has been commer-cially available in crystalline formsince 1954.

19

History

1831 Wackenroder isolates the orange-yellow pigment in carrots andcoins the term 'carotene'.

1847 Zeise provides a more detailed description of carotene.

1866 Carotene is classified as a hydrocarbon by Arnaud and co-workers.

1887 Arnaud describes the widespread presence of carotenes inplants.

1907 Willstatter and Mieg establish the molecular formula for carotene,a molecule consisting of 40 carbon and 56 hydrogen atoms.

1914 Palmer and Eckles discover the presence of carotene andxanthophylls in human blood plasma.

1919 Steenbock (University of Wisconsin) suggests a relationshipbetween yellow plant pigments (beta-carotene) and vitamin A.

1929 Moore demonstrates that beta-carotene is converted into thecolourless form of vitamin A in the liver.

1931 Karrer and collaborators (Switzerland) determine the structuresof beta-carotene and vitamin A.

1939 Wagner and coworkers suggest that the conversion of beta-carotene into vitamin A occurs within the intestinal mucosa.

1950 Isler and colleagues develop a method for synthesising beta-carotene.

1966 Beta-carotene is found acceptable for use in foods by the JointFAO/WHO Expert Committee on Food Additives.

1972 Specifications for beta-carotene use in foods is established bythe U.S. Food Chemicals Codex.

1979 Carotene is established as 'GRAS', which means that the ingre-dient is 'Generally Recognised As Safe' and can be used as adietary supplement or in food fortification.

1981-82 Beta-carotene/carotenoids are recognised as important factors(independent of their provitamin A activity) in potentially reducingthe risk of certain cancers. R. Doll and R. Peto: “Can DietaryBeta-carotene Materially Reduce Human Cancer Rates?” (in:Nature, 1981; 290: 201-208) R. Shekelle et al: “Dietary Vitamin Aand Risk of Cancer in the Western Electric Study” (in: Lancet,1981: 1185-1190) “Diet, Nutrition and Cancer” (1982): Review ofthe U.S. National Academy of Sciences showing that intake ofcarotenoid-rich foods is associated with reduced risk of certaincancers.

20

1982 Krinsky and Deneke show the interaction between oxygen andoxyradicals using carotenoids.

1983-84 The US National Cancer Institute (NCI) launches large-scaleclinical intervention trials using beta-carotene supplements aloneand in combination with other nutrients.

1984 Beta-carotene is demonstrated to be an effective antioxidant invitro.

1988 Due to the large number of epidemiological studies that demon-strate the potential reduction of cancer incidence with increasedconsumption of dietary beta-carotene, the US National CancerInstitute (NCI) issues dietary guidelines advising Americans toinclude a variety of vegetables and fruits in their daily diet.

1993-94 Availability of results from several large-scale clinical interventiontrials using beta-carotene alone or in various other combinations.

1997 Evidence indicates that beta-carotene acts synergistically withvitamins C and E.

1999 The Women´s Health Study shows no increased risk of lungcancer for woman receiving 50 mg beta-carotene on alternatedays.

2004 Results from the French SU.VI.MAX study indicate that a com-bination of antioxidant vitamins (C, E and beta-carotene) andminerals lowers total cancer incidence and all-cause mortality inmen.

Paul Karrer

Otto Isler

21

Vitamin D

SynonymsCalciferol; antirachitic factor; “sunshine” vitamin

ChemistryVitamin D is a generic term and indicates a molecule of the general struc-ture shown for rings A, B, C, and D with differing side chain structures. TheA, B, C, and D ring structure is derived from the cyclopentanoperhydro-phenanthrene ring structure for steroids. Technically, vitamin D is classifiedas a seco-steroid. Seco-steroids are those in which one of the rings hasbeen broken; in vitamin D, the 9,10 carbon-carbon bond of ring B is broken.

CH2

CH3

OH

H

H

H3CH

H

CH3

CH3

Molecular formula of vitamin D3 (cholecalciferol)

Vitamin D crystals in polarised light

22

IntroductionVitamin D is the general name givento a group of fat-soluble compoundsthat are essential for maintaining themineral balance in the body. Thechemical structure of vitamin D wasidentified in the 1930s. The mainforms are vitamin D2 (ergocalciferol:found in plants, yeasts and fungi)and vitamin D3 (cholecalciferol: ofanimal origin).

As cholecalciferol is synthesised inthe skin by the action of ultravioletlight on 7-dehydrocholesterol, acholesterol derivative, vitamin Ddoes not fit the classical definition ofa vitamin. Nevertheless, because ofthe numerous factors that influenceits synthesis, such as latitude, sea-son, air pollution, area of skinexposed, pigmentation, age, etc.,vitamin D is recognized as an essen-tial dietary nutrient.

Functions

Following absorption or endogenoussynthesis, the vitamin has to bemetabolised before it can perform itsbiological functions. Calciferol istransformed in the liver to 25-hydroxycholecalciferol (25(OH)D,calcidiol). This is the major circulat-ing form, which is metabolised in thekidney to the active forms asrequired. The most important ofthese is 1,25-dihydroxy-cholecalcif-erol (1,25(OH)2D, calcitriol) becauseit is responsible for most of the bio-logical functions. The formation of1,25(OH)2D, which is considered ahormone, is strictly controlledaccording to the body's calciumneeds. The main controlling factorsare the existing levels of 1,25(OH)2Ditself and the blood level of parathy-roid hormone, calcium and phos-phorus.To perform its biological functions,1,25(OH)2D, like other hormones,binds to a specific nuclear receptor

(vitamin D receptor, VDR). Uponinteraction with this receptor,1,25(OH)2D regulates more than 50genes in a wide variety of tissues.Vitamin D is essential for the controlof normal calcium and phosphateblood levels. It is known to berequired for the absorption of cal-cium and phosphate in the smallintestine, their mobilisation from thebones, and their reabsorption in thekidneys. Through these three func-tions it plays an important role forthe proper functioning of muscles,nerves and blood clotting and fornormal bone formation and minerali-sation.

It has been suggested that vitamin Dalso plays an important role in con-trolling cell proliferation and differen-tiation, immune responses andinsulin secretion.

Main functions in a nutshell:• Regulation of calcium and phos-

phate blood levels • Bone mineralisation• Control of cell proliferation and

differentiation• Modulation of immune system

Dietary sources

Vitamin D is found only in a fewfoods. The richest natural sources ofvitamin D are fish liver oils and salt-water fish such as sardines, herring,salmon and mackerel. Eggs, meat,milk and butter also contain smallamounts. Plants are poor sources,with fruit and nuts containing novitamin D at all. The amount of vita-min D in human milk is insufficient tocover infant needs.


Absorption of dietary vitamin D takesplace in the upper part of the smallintestine with the aid of bile salts. Itis incorporated into the chylomicronfraction and absorbed through thelymphatic system. Vitamin D isstored in adipose tissue. It has to bemetabolised to become active.

Measurement

Vitamin D status is best determinedby the serum 25(OH)D concentrationbecause this reflects dietary sourcesas well as vitamin D production byUV light in the skin. Usual serum25(OH)D values are between 25 and130 nmol/L depending on geograph-ic location.1 µg vitamin D is equivalent to 40 IU(international unit).

Stability

Vitamin D is relatively stable infoods. Storage, processing andcooking have little effect on its activ-ity, although in fortified milk up to40% of the vitamin D added may belost as a result of exposure to light.

Food Vitamin D (µg/100g)

Herring 25

Salmon 16

Sardines 11

Mackerel 4

Egg 2.9

Butter 1.2

Milk (whole) 0.07

Vitamin D content of foods


23

InteractionsPositive interactionsWomen taking oral contraceptiveshave been found to have slightly ele-vated blood levels of 1,25(OH)2D.

Negative interactionsCholestyramine (a resin used to stopreabsorption of bile salts) and laxa-tives based on mineral oil inhibit theabsorption of vitamin D from theintestine. Corticosteroid hormones,anticonvulsant drugs and alcoholcan affect the absorption of cal-cium by reducing the response to vitamin D.Animal studies also suggest thatanticonvulsant drugs stimulateenzymes in the liver, resulting in anincreased breakdown and excretionof the vitamin.

Deficiency

Among the first symptoms of mar-ginal vitamin D deficiency arereduced serum levels of calcium andan increase in parathyroid hormone(PTH) production. Serum alkalinephosphatase is elevated in vitamin Ddeficiency states. This can beaccompanied by muscle weaknessand tetany, as well as an increasedrisk of infection. Children may showunspecific symptoms, such as rest-lessness, irritabil ity, excessivesweating and impaired appetite.Marginal hypovitaminosis D maycontribute to bone brittleness in theelderly. Vitamin D deficiency canalso cause hearing loss.The most widely recognised mani-festations of severe vitamin D defi-ciency are rickets in children andosteomalacia in adults. Both arecharacterised by loss of mineral fromthe bones. This results in skeletaldeformities such as bowed legs inchildren. The ends of the long bonesin both the arms and legs areaffected, and their growth may beretarded. Rickets also results in

inadequate mineralisation of toothenamel and dentin.Osteoporosis, a disorder of olderage in which there is loss of bone,not just demineralisation, has alsobeen associated with less obviousstates of deficiency.

Groups at risk of deficiency:• Infants who are exclusively breast

fed are at high risk of vitamin Ddeficiency, because human milk isa poor source of vitamin D. Inaddition, in premature and low-birth-weight infants, liver and kid-ney function may be inadequatefor optimal vitamin D metabolism.

• The elderly have a reduced capac-ity to synthesise vitamin D in theskin by exposure to sunlight.

• People with diseases affecting theliver, kidneys, the thyroid gland orfat absorption, as well as vegetari-ans, alcoholics and epileptics onlong-term anticonvulsant therapyhave a greater risk of deficiency,as do people who are house-bound.

• Dark-skinned people produce lessvitamin D from sunlight and are atrisk of deficiency when living farfrom the equator.

• Populations living at latitudes ofaround 40 degrees north or southare exposed to insufficient levelsof sunlight to cover vitamin Drequirements through endogenousproduction, especially during win-ter months.

Hereditary vitamin D-dependentrickets (type I and II):These rare forms of rickets occurin spite of an adequate supply ofvitamin D. These are inheritedforms in which the formation orutil isation of 1,25(OH)2D isimpaired.

Diseaseprevention andtherapeutic use

In the treatment of rickets, a dailydose of 40 µg (1,600 IU) vitamin Dusually results in normal plasmaconcentrations of calcium and phos-phorus within 10 days. The dose canbe reduced gradually to 10 µg (400IU) per day after one month oftherapy.Vitamin D analogues are used in thetreatment of psoriasis.Vitamin D is discussed as a preven-tion factor for a number of diseases.Results from epidemiological studiesand evidence from animal modelssuggest that the risk of severalautoimmune diseases (multiple scle-rosis, insulin-dependent diabetesmellitus, rheumatoid arthritis) maybe decreased by adequate vitamin Dintake.Vitamin D plays an important role inthe prevention of osteoporosisbecause vitamin D insufficiency canbe an important contributing factorin this disease. A prospective studyamong 72,000 postmenopausalwomen over 18 years indicated thatwomen consuming at least 600 IUvitamin D/day from food plus sup-plements had a 37% lower risk ofhip fracture. Evidence from mostclinical trials suggests that vitamin Dsupplementation slows bone densitylosses and decreases the risk ofosteoporotic fracture in men andwomen. Various surveys and studies suggestthat poor vitamin D intake or statusis associated with an increased riskof colon, breast and prostate cancer.

24


Establishing an RDA for vitamin D isdifficult because vitamin D can beendogenously produced in the bodythrough exposure to sunlight.Healthy people regularly exposed tothe sun have no dietary requirementfor vitamin D, under appropriateconditions. As this is rarely the casein temperate zones, however, adietary supply is needed.In 1997, the Food and NutritionBoard based adequate intake levels(AI) on the assumption that no vita-min D is produced by UV light in theskin. An AI of 5 µg (200 IU)/day isrecommended for infants, childrenand adults (ages 19-50 years). Forthe elderly, higher intakes are rec-ommended to maintain normal calci-um metabolism and maximise bonehealth. In other countries, adult rec-ommendations range from 2.5 µg(100 IU) to 10 µg (400 IU).

SafetyHypervitaminosis D is a potentiallyserious problem as it can causepermanent kidney damage, growthretardation, calcification of softtissues and death. Mild symptoms ofintoxication are nausea, weakness,constipation and irritability. In gener-al, the toxic dose for adults isaround 1.25 mg (50,000 IU) per day.However, certain individuals have anincreased sensitivity to vitamin Dand present with toxic symptomsafter 50 µg (2,000 IU) per day.Hypervitaminosis D is not associatedwith overexposure to the sunbecause a regulating mechanismprevents overproduction of vitaminD.

The Food and Nutrition Board (FNB)and the EU Scientific Committee onFood have set the tolerable upperintake level (UL) for vitamin D at 50µg/day for adolescents and adults.

Supplements andfood fortificationMonopreparations of vitamin D andrelated compounds are available astablets, capsules, oily solutions andinjections. Vitamin D is also incorpo-rated in combinations with vitamin A,calcium, and in multivitamins.In many countries, milk and milkproducts, margarine and vegetableoils fortified with vitamin D serve asa major dietary source of the vita-min.


Cholecalciferol is produced com-mercially by the action of ultravioletlight on 7-dehydrocholesterol, whichis obtained from cholesterol by vari-ous methods. Ergocalciferol is pro-duced in a similar manner fromergosterol, which is extracted fromyeast. Starting material for the pro-duction of caIcitriol is the cholesterolderivative pregnenolone.


RDA*

Infants 5 µg (AI)

Children 1-18 years 5 µg (AI)

Males 19-50 years 5 µg (AI)

Females 19-50 years 5 µg (AI)

Males 51- 70 years 10 µg (AI)

Females 51-70 years 10 µg (AI)

Males . 70 years 15 µg (AI)

Females . 70 years 15 µg (AI)

Pregnancy 5 µg (AI)

Lactation 5 µg (AI)












History

1645 Whistler writes the first scientific description of rickets.

1865 In his textbook on clinical medicine, Trousseau recommends codliver oil as treatment for rickets. He also recognises the impor-tance of sunlight and identifies osteomalacia as the adult form ofrickets.

1919 Mellanby proposes that rickets is due to the absence of a fat-soluble dietary factor.

1922 McCollum and coworkers establish the distinction betweenvitamin A and the antirachitic factor.

1925 McCollum and coworkers name the antirachitic factor vitamin D.Hess and Weinstock show that a factor with antirachitic activityis produced in the skin by ultraviolet irradiation.

1936 Windaus identifies the structure of vitamin D in cod liver oil.

1937 Schenck obtains crystallised vitamin D3 by activation of 7-dehy-dro-cholesterol.

1968 Haussler and colleagues report the presence of an activemetabolite of vitamin D in the intestinal mucosa of chicks.

1969 Haussler and Norman discover calcitriol receptors in chickintestine.

1970 Fraser and Kodicek discover that calcitriol is produced in thekidney.

1971 Norman and coworkers identify the structure of calcitriol.

1973 Fraser and associates discover the presence of an inborn errorof vitamin D metabolism that produces rickets resistant tovitamin D therapy.

1978 De Luca's group discovers a second form of vitamin D-resistantrickets (Type II).

1981 Abe and colleagues in Japan demonstrate that calcitriol isinvolved in the differentiation of bone-marrow cells.

1983 Provvedini and colleagues demonstrate the presence of calcitriolreceptors in human leukocytes.

1984 The same group presents evidence that calcitriol has a regulato-ry role in immune function.

1986 Morimoto and associates suggest that calcitriol may be useful inthe treatment of psoriasis.

25

26

Elmer V. McCollum

Adolf Windaus

Sir Edward Mellanby

1989 Baker and associates show that the vitamin D receptor belongsto the steroid-receptor gene family.

1994 The U.S. Food and Drug Administration approves a vitamin D-based topical treatment for psoriasis, called calcipotriol.

2003 A prospective study from Feskanich and coworkers among72,000 postmenopausal women in the U.S. over 18 years indi-cated that women consuming at least 600 IU vitamin D/day fromfood plus supplements had a 37% lower risk of hip fracture.

2006 Researchers from the Harvard School of Public Health examinedcancer incidence and vitamin D exposure in over 47,000 men inthe Health Professionals Follow-Up Study. They found that a highlevel of vitamin D (~1500 IU daily) was associated with a 17%reduction in all cancer incidences and a 29% reduction in totalcancer mortality with even stronger effects for digestive-systemcancers.

27

Vitamin E

SynonymsTocopherol

ChemistryA group of compounds composed of a substituted chromanol ring with aC16 side chain saturated in tocopherols, with 3 double bonds intocotrienols.

CH3

CH3

CH3H3C

HO

OCH3 CH3

CH3

CH3

Molecular formula of a-tocopherol

Vitamin E crystals in polarised light

28

Introduction

The term vitamin E covers eight fat-soluble compounds found in nature.Four of them are called tocopherolsand the other four tocotrienols. Theyare identified by the prefixes a, b, gand d. a-Tocopherol is the mostcommon and biologically the mostactive of these naturally occurringforms of vitamin E. Natural toco-pherols occur in RRR-configurationonly (RRR-a-tocopherol was former-ly designated as d-a-tocopherol).The chemical synthesis of a-toco-pherol results in a mixture of eightdifferent stereoisomeric forms whichis called all-rac-a-tocopherol (or dl-a-tocopherol). The biological activityof the synthetic form is lower thanthat of the natural form.The name tocopherol derives fromthe Greek words tocos, meaningchildbirth, and pherein, meaning tobring forth. The name was coined tohighlight its essential role in thereproduction of various animalspecies.The ending -ol identifies the sub-stance as being an alcohol.The importance of vitamin E inhumans was not accepted until fair-ly recently. Because its deficiency isnot manifested by a well-recognised,widespread vitamin deficiency dis-ease such as scurvy (vitamin Cdeficiency) or rickets (vitamin D defi-ciency), science only began torecognise the importance of vitaminE at a relatively late stage.

Functions

The major biological function of vita-min E is that of a lipid soluble antiox-idant preventing the propagation offree-radical reactions. Free radicalsare formed in normal metabolicprocesses and upon exposure toexogenous toxic agents (e.g. ciga-rette smoke, pollutants). Vitamin E islocated within the cellular mem-

branes. It protects polyunsaturatedfatty acids (PUFAs) and other com-ponents of cellular membranes fromoxidation by free radicals. Apartfrom maintaining the integrity of thecell membranes in the human body,it also protects low density lipo-protein (LDL) from oxidation. Recently, non-antioxidant functionsof a-tocopherol have been identi-fied.a-Tocopherol inhibits protein kinaseC activity, which is involved in cellproliferation and differentiation.Vitamin E inhibits platelet aggrega-tion and enhances vasodilation.Vitamin E enrichment of endothelialcells downregulates the expressionof cell adhesion molecules, therebydecreasing the adhesion of bloodcell components to the endothelium.

Main functions in a nutshell:• Major fat soluble antioxidant of

the body• Non-antioxidant functions in cell

signalling, gene expression andregulation of other cell functions

Dietary sources

Vegetable oils (olive, soya beans,palm, corn, safflower, sunflower,etc.), nuts, whole grains and wheatgerm are the most importantsources of vitamin E. Other sources

are seeds and green leafy vegeta-bles. The vitamin E content of veg-etables, fruits, dairy products, fishand meat is relatively low.The vitamin E content in foods isoften reported as a-tocopherolequivalents (a-TE). This term wasestablished to account for the differ-ences in biological activity of thevarious forms of vitamin E. 1 mg ofa-tocopherol is equivalent to 1 TE.Other tocopherols and tocotrienolsin the diet are assigned the followingvalues: 1 mg b-tocopherol = 0.5 TE;1 mg g-tocopherol = 0.1 TE; 1 mgd-tocopherol = 0.03 TE; 1 mg a-tocotrienol = 0.3 TE; 1 mg b-tocotrienol = 0.05 TE.

Food Vitamin E (mg a-TE/100g)

Wheat germ oil 174

Sunflower oil 63

Hazelnut 26

Rape seed oil 23

Soya bean oil 17

Olive oil 12

Peanuts 11

Walnuts 6

Butter 2

Spinach 1.4

Tomatoes 0.8

Apples 0.5

Milk (whole) 0.14

Vitamin E content of foods


29


Vitamin E is absorbed together withlipids in the small intestine, depend-ing on adequate pancreatic functionand biliary secretion. Tocopherolesters which are present in foodsupplements and processed foodare hydrolysed before absorption.Vitamin E is incorporated intochylomicrons and transported viathe lymphatic system to the liver.a-Tocopherol is the vitamin E formthat predominates in blood and tis-sue. This is due to the action of aliver protein (a-tocopherol transferprotein) preferentially incorporatinga-tocopherol into the lipoproteinswhich deliver it to the different tis-sues. Vitamin E is found in mosthuman body tissues. The highestvitamin E contents are found in theadipose tissue, liver and muscles.The pool of vitamin E in the plasma,liver, kidneys and spleen turns overrapidly, whereas turnover of the con-tent of adipose tissue is slow.

Measurement

Normal a-tocopherol concentrationsin plasma measured by high per-formance liquid chromatographyrange from 12-45 µM (0.5-2 mg/100ml). Plasma a-tocopherol concen-trations of <11.6 µM, the level atwhich erythrocyte haemolysesoccurs, indicate poor vitamin Enutritional status. Since plasmalevels of a-tocopherol correlate withcholesterol levels, the a-tocopherolconcentration is often indicated asa-tocopherol-cholesterol ratio.Vitamin E content is generallyexpressed by biological activity,using the scale of International Units(IU). According to this system, 1 mgof RRR-a-tocopherol, biologicallythe most active of the naturallyoccurring forms of vitamin E, isequivalent to 1.49 IU vitamin E. The

biological activity of 1 mg of all-rac-a-tocopheryl acetate, the synthe-sised form of vitamin E commonlyused in food enrichment, is equiva-lent to 1 IU. Recently, the unit of a-tocopherol equivalent was estab-lished (see: Dietary sources).

Stability

Light, oxygen and heat, detrimentalfactors encountered in long storageof foodstuffs and food processing,lower the vitamin E content of food.In some foods it may decrease by asmuch as 50% after only two weeks'storage at room temperature. To alarge extent, frying destroys the vita-min E in vegetable oils.Esters of a-tocopherol (a-toco-pheryl acetate and a-tocopherylsuccinate) are used for supplementsbecause they are more resistant tooxidation during storage.

Interactions

Positive interactionsThe presence of other antioxidants,such as vitamin C and beta-carotene, supports the antioxidative,protective action of vitamin E; thesame is true of the mineral selenium.

Negative interactionsWhen taken at the same time, ironreduces the availability of vitamin Eto the body; this is especially criticalin the case of anaemic newborns.The requirement for vitamin E isrelated to the amount of polyunsatu-rated fatty acids consumed in thediet. The higher the amount ofPUFAs, the more vitamin E isrequired.Vitamin K deficiency may be exacer-bated by vitamin E, thereby affectingblood coagulation.Various medications decreaseabsorption of vitamin E (e.g.,cholestyramine, colestipol, isoniazid).

30

DeficiencyBecause depletion of vitamin E tis-sue stores takes a very long time, noovert clinical deficiency symptomshave been noted in otherwisehealthy adults. Symptoms of vitaminE deficiency are seen in patientswith fat malabsorption syndromes orliver disease, in individuals withgenetic defects affecting the a-toco-pherol transfer protein and in new-born infants, particularly prematureinfants.Vitamin E deficiency results in neuro-logical symptoms (neuropathy),myopathy (muscle weakness) andpigmented retinopathy. Early diag-nostic signs are leakage of muscleenzymes, increased plasma levels oflipid peroxidation products andincreased haemolysis of erythro-cytes (red blood cells). In prematureinfants, vitamin E deficiency is asso-ciated with haemolytic anaemia,intraventricular haemorrhage andretrolental fibroplasia.


Research studies suggest that vita-min E has numerous health benefits.Vitamin E is thought to play a role inpreventing atherosclerosis and car-diovascular diseases (heart diseaseand stroke) due to its effects on anumber of steps in the developmentof atherosclerosis (e.g. inhibition ofLDL oxidation, inhibition of smoothmuscle cell proliferation, inhibition ofplatelet adhesion, aggregation andplatelet release reaction). Recent studies suggest that vitaminE enhances immunity in the elderly,and that supplementation with vita-min E lowers the risk of contractingan upper respiratory tract infection,particularly the common cold. Researchers are investigating the

prophylactic role of vitamin E in pro-tecting against exogenous pollutantsand lowering the risk of cancer andof cataracts.Vitamin E in combination with vita-min C may protect the body fromoxidative stress caused by extremesports (e.g. ultra marathon running).A role of vitamin E supplementationin the treatment of neurodegenera-tive diseases (Alzheimer´s disease,amyotrophic lateral sclerosis) is alsounder investigation.

RecommendedDietary Allowance(RDA)The recommended daily intake ofvitamin E varies according to age,sex and criteria applied in individualcountries. In the USA, the RDA foradults is 15 mg RRR-a-toco-pherol/day (FNB, 2000). In Europe,adult recommendations range from4 to 15 mg a-TE/day for men andfrom 3 to 12 mg a-TE/day forwomen.The RDA for vitamin E of 15 mg can-

not easily be acquired even with thebest nutritional intentions, yet mostresearch studies show that optimalintake levels associated with healthbenefits tend to be high. Vitamin Eintake should also be adapted tothat of PUFA, which influences therequirement for this vitamin. The ECScientific Committee on Foods (SCF)has suggested a consumption ratioof 0.4 mg a-TE per gram of PUFA.

Safety

Vitamin E has low toxicity. Afterreviewing more than 300 scientificstudies, the US-based Institute ofMedicine (IOM) concluded that vita-min E is safe for chronic use even atdoses of up to 1000 mg per day. Arecently published meta-analysissuggested that taking more than400 IU of vitamin E per day broughta weekly increase in the risk of all-cause mortality. However, much ofthe research was done in patients athigh risk of a chronic disease andthese findings may not be generalis-able to healthy adults. Many humanlong-term studies with higher doses


RDA*

Infants # 6 months 4 mg (6 IU)(AI)

Infants 7-12 months 5 mg (7.5 IU)(AI)

Children 1-3 years 6 mg (9 IU)

Children 4-8 years 7 mg (10.5 IU)

Children 9-13 years 11 mg (16.5 IU)

Males $ 14 years 15 mg (22.5 IU)

Females $ 14 years 15 mg (22.5 IU)

Pregnancy 15 mg (22.5 IU)

Lactation 19 mg (28.5 IU)












31

of vitamin E have not reported anyadverse effects, and it has beenconcluded that vitamin E intakes ofup to 1600 IU (1073 mg RRR-a-tocopherol) are safe for most adults.The Antioxidant Panel of the Foodand Nutrition Board (FNB, 2000) hasset the UL (tolerable upper intakelevel) for adults at 1000 mg/day ofany form of supplemental a-toco-pherol. In 2003 the EC ScientificCommittee on Foods (SCF) estab-lished the UL of 300 mg a-TE foradults. Also in 2003, the UK Expertgroup on Vitamins and Minerals(EVM; 2003) set the UL at 540 mga-TE for supplemental vitamin E.Pharmacologic doses of vitamin Emay increase the risk of bleeding inpatients treated with anticoagulants.Patients on anticoagulant therapy orthose anticipating surgery shouldavoid high levels of vitamin E.

Supplements, food fortifica-tions and otherapplications

Vitamin E is available insoft gelatine capsules, or aschewable or effervescenttablets, and is found in mostmultivitamin supplements.The most common fortifiedfoods are soft drinks andcereals.

The all-rac-a-toco-pherol form of vitamin Eis widely used as anantioxidant in stabilising edibleoils, fats and fat-containing foodproducts.Research has shown thatvitamin E in combinationwith vitamin C reduces theformation of nitrosamines (a provencarcinogen in animals) in baconmore effectively than vitamin Calone.

Vitamin E has been used topically asan anti-inflammatory agent, toenhance skin moisturisation and toprevent cell damage by UV light.In pharmaceutical products toco-pherol is used, for example, to sta-bilise syrups, aromatic components,and vitamin A or provitamin A com-ponents.a-Tocopherol is used as an antioxi-dant in plastics, technical oils andgreases, and in the purified, so-called white oils, employed in cos-metics and pharmaceuticals.


Vitamin E derived from naturalsources is obtained by moleculardistil lation and, in most cases,subsequent methylation and esterifi-cation of edible vegetable oil prod-ucts. Synthetic vitamin E is pro-duced from fossil plant material bycondensation of trimethylhydro-quinone with isophytol.

Erhard Fernholz

Paul Karrer

Katherine Scott Bishop

32

Herbert Evans

History

1911 Hart and coworkers publish the first report of a suspected “anti-sterility factor” in animals.

1920 Matthill and Conklin observe reproductive anomalies in rats fedon special milk diets.

1922 Vitamin E is discovered by Evans and Scott Bishop.

1936 Evans and coworkers isolate what turns out to be a-tocopherolin its pure form from wheat germ oil.

1938 Fernholz provides the structural formula of vitamin E and Nobellaureate Karrer synthesises dl-a-tocopherol.

1945 Dam and coworkers discover peroxides in the fat tissue of ani-mals fed on vitamin E-deficient diets. The first antioxidant theoryof vitamin E activity is proposed.

1962 Tappel proposes that vitamin E acts as an in vivo antioxidant toprotect cell lipids from free radicals.

1968 The Food and Nutrition Board of the US National ResearchCouncil recognises vitamin E as an essential nutrient for humans.

1974 Fahrenholtz proposes singlet oxygen quenching abilities of a-tocopherol.

1977 Human vitamin E deficiency syndromes are described.

1980 Walton and Packer propose that vitamin E may prevent thegeneration of potentially carcinogenic oxidative products ofunsaturated fatty acids.

1980 McKay and King suggest that vitamin E functions as an antioxi-dant located primarily in the cell membrane.

1980s Vitamin E is demonstrated to be the major lipid-soluble antioxi-dant protecting cell membranes from peroxidation. Vitamin E isshown to stabilise the superoxide and hydroxyl free radicals.

1990 Effectiveness of vitamin E in inhibiting LDL (low density lipopro-tein) oxidation is shown.

1990 Kaiser and coworkers elucidate the singlet oxygen quenchingcapability of vitamin E.

1991 Azzi and coworkers describe an inhibitory effect of a-tocopherolon the proliferation of vascular smooth muscle cells and proteinkinase C activity.

2004 Barella and coworkers demonstrate that vitamin E regulates geneexpression in the liver and the testes of rats.

33

Vitamin K

SynonymsPhylloquinone, menaquinone

ChemistryCompounds with vitamin K activity are 3-substituted 2-methyl-1,4-naphtho-quinones. Phylloquinone contains a phytyl group, whereas menaquinonescontain a polyisoprenyl side chain with 6 to 13 isoprenyl units at the 3-posi-tion.

CH3

O

O CH3 CH3 CH3

CH3

CH3

Molecular formula of vitamin K1 (phylloquinone)

Vitamin K crystals in polarised light

34

Introduction

In 1929 Henrik Dam observed thatchicks fed on fat-free diets devel-oped haemorrhages and startedbleeding. In 1935 he proposed thatthe antihaemorrhagic substance wasa new fat-soluble vitamin, which hecalled vitamin K (after the first letterof the German word “Koagulation”).Vitamin K is indeed fat-soluble, andit occurs naturally in two forms: vita-min K1 (phylloquinone) is found inplants; vitamin K2 is the term for agroup of compounds called mena-quinones (MK-n, n being the numberof isoprenyl units in the side chain ofthe molecule) which are synthesisedby bacteria in the intestinal tract ofhumans and various animals.Vitamin K3 (menadione) is a synthet-ic compound that can be convertedto K1 in the intestinal tract. It is onlyused in animal nutrition.

Functions

Vitamin K is essential for the synthe-sis of the biologically active forms ofa range of proteins called vitamin K-dependent proteins. Vitamin K par-ticipates in the conversion of gluta-mate residues of these proteins tog-carboxylglutamate residues byaddition of a carboxyl-group (car-boxylation).

In the absence of vitamin K, car-boxylation of these proteins isincomplete, and they are secreted inplasma in various so called under-carboxylated forms, which are bio-logically inactive.Vitamin K is also essential for thefunctioning of several proteinsinvolved in blood coagulation (clot-ting), a mechanism that preventsbleeding to death from cuts andwounds, as well as internal bleeding.

Vitamin K-dependent proteins:• Prothrombin (factor II), factors VII,

IX, and X, and proteins C, S and Zare proteins that are involved inthe regulation of blood coagula-tion. They are synthesised in theliver. Protein S has also beendetected in bone.

• The vitamin K-dependent proteinsosteocalcin and MGP (matrix Gla-protein) have been found in bone.Osteocalcin is thought to be relat-ed to bone mineralisation. MatrixGla-protein is present in bone,cartilage and vessel walls and hasrecently been established as aninhibitor of calcification. The role ofprotein S in bone metabolism isnot clear.

• Recently, several other vitamin K-dependent proteins have beenidentified.

Main functions in a nutshell:• Coenzyme for a vitamin

K-dependent carboxylase• Blood coagulation• Bone metabolism

Dietary sources

The best dietary sources of vitaminK1 are green leafy vegetables suchas spinach, broccoli, Brusselssprouts, cabbage and lettuce. Otherrich sources are certain vegetableoils. Good sources include oats,potatoes, tomatoes, asparagus andbutter. Lower levels are found inbeef, pork, ham, milk, carrots, corn,most fruits and many other vegeta-bles.An important source of vitamin K2 isthe bacterial flora in the anterior partof the gut – the jejunum and ileum.

Food Vitamin K (µg/100g)

Spinach 305

Brussels sprouts 236

Broccoli 155

Rape seed oil 150

Soya bean oil 138

Lettuce 109

Cabbage 66

Asparagus 39

Olive oil 33

Butter 7

Vitamin K content of foods


Absorptionand body storesVitamin K is absorbed from thejejunum and ileum. As with other fat-soluble vitamins, absorptiondepends on the presence of bile andpancreatic juices and is enhancedby dietary fat. Although the liver isthe main storage site, vitamin K isalso found in extrahepatic tissues,e.g. bone and heart. Liver storesconsist of about 10% phylloqui-nones and 90% menaquinones.Compared with that of other fat-sol-uble vitamins, the total body pool ofvitamin K is small and turnover ofvitamin K in the liver is rapid. Thebody recycles vitamin K in a processcalled the vitamin K cycle, allowingthe vitamin to function in the g-car-boxylation of proteins many timesover.Although the liver containsmenaquinones synthesised by intes-tinal bacteria, the absorption ofmenaquinones and their contributionto the human vitamin K requirementhave not yet been fully elucidated.

Measurement

Plasma vitamin K concentration ismeasured by high performance liq-uid chromatography. The normalrange of plasma vitamin K in adultsis 0.2-3.2 ng/ml. Levels below 0.5ng/ml have been associated withimpaired blood-clotting functions.However, the value of measuringplasma vitamin K concentration toassess vitamin K status is limitedbecause it responds to changes in

dietary intake within 24 hours.Overt vitamin K deficiency re-

sults in impaired bloodclotting, usually demon-strated by laboratory

tests that measure clot-ting time.Plasma concentration of one of

the vitamin K-dependent blood-clotting factors – prothrombin, factorVII, factor IX or factor X – is meas-ured to assess an inadequate intakeof vitamin K. The normal range ofplasma prothrombin concentration isfrom 80 to 120 µg/ml.

Recently, other parameters forassessing vitamin K status havebeen studied, e.g. measurements ofundercarboxylated prothrombin andundercarboxylated osteocalcin inboth normal and pathological condi-tions.

Stability

Vitamin K compounds are moderate-ly stable to heat and reducingagents, but are sensitive to acid,alkali, light and oxidising agents.

Interactions

Negative interactions• Coumarin anticoagulants (such as

warfarin), salicylates and certainantibiotics act as vitamin K antag-onists.

• Very high dietary or supplementalintakes of vitamin K may inhibit theanticoagulant effect of vitamin Kantagonists (e.g. warfarin).

• High doses of vitamins A and Ehave been shown to interfere withvitamin K and precipitate deficien-cy states.

• Absorption of vitamin K may bedecreased by mineral oil, bile acidsequestrants (cholestyramine,colestipol) and orlistat (weight lossmedication).

Deficiency

Vitamin K deficiency is uncommon inhealthy adults but occurs in individ-uals with gastrointestinal disorders,fat malabsorption or liver disease, orafter prolonged antibiotic therapycoupled with compromised dietaryintake. Impaired blood clotting is theclinical symptom of vitamin K defi-ciency, which is demonstrated bymeasuring clotting time. In severecases, bleeding occurs. Adults atrisk of vitamin K deficiency alsoinclude patients taking anticoagulantdrugs which are vitamin K antago-nists.

Newborn infants have a well estab-lished risk of vitamin K deficiency,which may result in fatal intracranialhaemorrhage (bleeding within theskull) in the first weeks of l ife.Breast-fed infants in particular havea low vitamin K status because pla-cental transfer of vitamin K is poorand human milk contains low levelsof vitamin K. The concentrations ofplasma clotting factors are low ininfants due to immaturity of the liver.Haemorrhagic disease in the new-born is a significant worldwide causeof infant morbidity and mortality.Therefore, in many countries vitaminK is routinely administered prophy-lactically to all newborns.

Diseaseprevention andtherapeutic usePhylloquinone is the preferred formof the vitamin for clinical use. It isused for intravenous and intramus-cular injections as a colloidal sus-pension, emulsion or aqueous sus-pension, and as a tablet for oral use.Vitamin K1 is used in the treatmentof hypoprothrombinemia (lowamounts of prothrombin), secondaryto factors limiting absorption or syn-

36

thesis of vitamin K. During opera-tions in which bleeding is expectedto be a problem, for example, in gall-bladder surgery, vitamin K1 is admin-istered.Anticoagulants inhibit vitamin Krecycling, which can be correctedrapidly and effectively by the admin-istration of vitamin K1.Vitamin K1 is often given to mothersbefore delivery and to newborninfants to protect against intracranialhaemorrhage.A putative role of vitamin K in osteo-porosis has been investigated sincevitamin K-dependent proteins havebeen discovered in bone. However,further investigations are required toresolve whether vitamin K is a signif-icant etiological component ofosteoporosis. A role for vitamin K inthe development of atherosclerosisis also under discussion, but studiessupporting this hypothesis are limit-ed and future research is recom-mended.

Recently, studies with cancer celllines and animal studies have indi-cated that a combination of vitaminC and vitamin K3 has antitumoractivity and inhibits the developmentof metastases.

RecommendedDaily Allowance(RDA)The US Food and Nutrition Board ofthe Institute of Medicine (2001) hasestablished an adequate intake (AI)level for adults based on reporteddietary intakes of vitamin K in appar-ently healthy population groups.Other health authorities have cometo similar conclusions.

Safety

Even when large amounts of vitaminK1 and K2 are ingested over anextended period, toxic manifesta-tions have not been observed.Therefore, the major health authori-ties have not established a tolerableupper level of intake (UL) for vitaminK. Allergic reactions have beenreported, however. Furthermore,administered menadione (K3) hasbeen known to cause haemolyticanaemia, jaundice and kernicterus (agrave form of jaundice in the new-born) and is no longer used for treat-ment of vitamin K deficiency.

Supplements, foodfortification andother applications

Supplements of vitamin K are availablealone in tablets and capsules, and alsoin multivitamin preparations.Infant formula products, beverages andcookies are fortified with vitamin K.Menadione salts are generally pre-ferred for farm animals because oftheir stability.


The procedure involves the use ofmonoester, menadiol and an acidcatalyst. Purification of the desiredproduct to remove unreactedreagents and side products occurseither at the quinol stage or afteroxidation.


RDA*

Infants , 6 months 2 µg (AI)

Infants 7-12 months 2.5 µg (AI)





Males . 19 years 120 µg (AI)

Females . 19 years 90 µg (AI)

Pregnancy 14-18 years 75 µg (AI)

Pregnancy . 19 years 90 µg (AI)

Lactation 14-18 years 75 µg (AI)

Lactation . 19 years 90 µg (AI)












Carl P. H. Dam

Edward A. Doisy

37

History

1929 A series of experiments by Dam results in the discovery ofvitamin K.

1931 A clotting defect is observed by McFarlane and coworkers.

1935 Dam proposes that the antihaemorrhagic vitamin in chicks is anew fat-soluble vitamin, which he calls vitamin K.

1936 Dam and associates succeed in preparing a crude plasmaprothrombin fraction, and demonstrate that its activity isdecreased when it is obtained from vitamin K-deficient chickplasma.

1939 Vitamin K1 is synthesised by Doisy and associates.

1940 Brikhous observes haemorrhagic conditions resulting frommalabsorption syndromes or starvation, and finds that haemor-rhagic disease of the newborn responds to vitamin K.

1943 Dam receives half of the Nobel prize for his discovery of vitaminK, the blood coagulation factor.

1943 Doisy receives half of the Nobel prize for his discovery of thechemical nature of vitamin K.

1974 The vitamin K-dependent step in prothrombin synthesis isdemonstrated by Stenflo and associates and Nelsestuen andcolleagues.

1975 Esmon discovers a vitamin K-dependent protein carboxylation inthe liver.

38

Vitamin C

SynonymsAscorbic acid, hexuronic acid, anti-scorbutic vitamin

ChemistryL-ascorbic acid (2,3-endiol-L-gulonic acid-g-lactone), dehydro-L-ascorbicacid (2-oxo-L-gulonic acid- g-lactone).

OH

O

CH2OH

O

H

OH OHH

Molecular formula of vitamin C

Vitamin C crystals in polarised light

39

Introduction

Vitamin C is water-soluble, andprobably the most famous of all thevitamins. Even before its discoveryin 1932, physicians recognised thatthere must be a compound in citrusfruits preventing scurvy, a diseasethat killed as many as 2 millionsailors between 1500 and 1800.Later researchers discovered thatman, other primates and the guineapig depend on external sources tocover their vitamin C requirements.Most other animals are able to syn-thesise vitamin C from glucose andgalactose in their body.

Functions

The most prominent role of vitamin Cis its immune stimulating effect,which is important for the defenceagainst infections such as commoncolds. It also acts as an inhibitor ofhistamine, a compound that isreleased during allergic reactions. Asa powerful antioxidant it can neu-tralise harmful free radicals and aidsin neutralising pollutants and toxins.Thus it is able to prevent the forma-tion of potentially carcinogenicnitrosamines in the stomach (due toconsumption of nitrite-containingfoods, such as smoked meat).Importantly, vitamin C is also able toregenerate other antioxidants suchas vitamin E. Vitamin C is requiredfor the synthesis of collagen, theintercellular “cement” substancewhich gives structure to muscles,vascular tissues, bones, tendonsand ligaments. Due to these func-tions vitamin C, especially in combi-nation with zinc, is important for thehealing of wounds. Vitamin C con-tributes to the health of teeth andgums, preventing haemorrhagingand bleeding. It also improves theabsorption of iron from the diet, andis needed for the metabolism of bileacids, which may have implications

for blood cholesterol levels and gall-stones. In addition, vitamin C playsan important role in the synthesis ofseveral important peptide hormonesand neurotransmitters and carnitine.Finally, vitamin C is also a crucialfactor in the eye's ability to deal withoxidative stress, and can delay theprogression of advanced age-relatedmacular degeneration (AMD) andvision-loss in combination with otherantioxidant vitamins and zinc.

Main functions in a nutshell:• Immune stimulation• Anti-allergic• Antioxidant• “Cement” for connective tissues• Wound healing• Teeth and gum health• Aids iron absorption• Eye health

Dietary sources

Vitamin C is widely distributed infruits and vegetables. Citrus fruits,blackcurrants, peppers, green veg-etables (e.g. broccoli, Brusselssprouts), and fruits like strawberries,guava, mango and kiwi are particu-larly rich sources. On a quantitybasis, the intake of potatoes, cab-

bage, spinach and tomatoes is alsoof importance. Depending on theseason, one medium-sized glass offreshly pressed orange juice (i.e. 100g) yields from 15 to 35 mg vitamin C.


Intestinal absorption of vitamin Cdepends on the amount of dietaryintake, decreasing with increasingintake levels. At an intake of 30 to180 milligrams, about 70% to 90% isabsorbed; about 50% of a singledose of 1 to 1.5 grams is absorbed;and only 16% of a single dose of 12grams is absorbed. Up to about 500milligrams are absorbed via a sodi-um-dependent active transportprocess, while at higher doses sim-ple diffusion occurs.

The storage capacity of water-solu-ble vitamins is generally low com-pared to that of fat-soluble ones.Humans have an average tissuestore of vitamin C of 20 mg/kg bodyweight. The highest concentration isfound in the pituitary gland (400mg/kg); other tissues of high con-centration are the adrenal glands,liver, brain and white blood cells(leukocytes).

Measurement

Vitamin C can be measured in theblood plasma and other body tis-sues by various techniques. Alsodipstick tests for estimation of vita-min C levels in the urine are avail-able. Less satisfying, however, is theevaluation of the analytical data con-cerning the true reflection of thebody status. Threshold values aredifficult to define and the subject ofcontroversial discussion. Typicalblood plasma levels are in the rangeof 20 to 100 µmol/L.

Food Vitamin C (mg/100g)

Acerolas 1600

Blackcurrants 200

Peppers 138

Broccoli 115

Fennel 95

Kiwis 71

Strawberries 64

Oranges 49

Vitamin C content of foods


40

StabilityVitamin C is sensitive to heat, lightand oxygen. In food it can be partlyor completely destroyed by longstorage or overcooking. Refrige-ration can substantially diminishvitamin C loss in food.

Modified from Oberbeil, Fit durchVitamine, Die neuen Wunderwaffen,Südwest Verlag GmbH & Co. KG,München 1993

Interactions

Positive interactionsThe presence of other antioxidants,such as vitamin E and beta-carotene, supports the protectiveantioxidant action of vitamin C.Other vitamins, such as those of theB-complex (particularly B6, B12, folicacid and pantothenic acid) andsome pharmacologically active sub-stances, as well as the naturallyoccurring compounds known asbioflavonoids, may have a spar-ing effect on vitamin C.

Negative interactionsDue to toxic compounds insmoke, the vitamin C require-ment for smokers is about 35mg/day higher than for non-smokers. Also several pharma-cologically active compounds,among them some anti-depres-

sants, diuretics, birth control pillsand aspirin, deplete the tissues ofvitamin C. This is also true of certainhabits, for example alcohol con-sumption.

Deficiency

Early symptoms of vitamin C defi-ciency are very general and couldalso indicate other diseases. Theyinclude fatigue, lassitude, loss ofappetite, drowsiness and insomnia,feeling run-down, irritabil ity, lowresistance to infections and petechi-ae (minor capillary bleeding). Severevitamin C deficiency leads to scurvy,

characterised by weakening of col-lagenous structures, resulting inwidespread capillary bleeding.Infantile scurvy causes bone malfor-mations. Bleeding gums and loosen-ing of the teeth are usually the earli-est signs of clinical deficiency.Haemorrhages under the skin causeextreme tenderness of extremitiesand pain during movement. If leftuntreated, gangrene and death mayensue. Nowadays this is rare indeveloped countries and can be pre-vented by a daily intake of about 10-15 mg of vitamin C. However, foroptimal physiological functioningmuch higher amounts are required.

The development of vitamin Cdeficiency can be caused by:• Inadequate storage and prepa-

ration of food• Gastrointestinal disturbances • Stress and exercise• Infections• Smoking• Diabetes• Pregnancy and lactation

Influence of storage and preparation on vitamin C loss in foods

Food Storage/Preparation Vitamin C Loss

Potatoes 1 month 50%

Fruit 1 month 20%

Apples 6-9 months 100%

Milk UHT 25%

Fruit Sterilisation 50%

Fruit Air drying 50-70%

Leafy vegetables Canning 48%

41


Dozens of prospective studies sug-gest that vitamin C plays a role inpreventing a variety of diseases. It isalso used to treat certain diseases inorthomolecular medicine. As thisnutrient is important for a variety ofdiseases, only a selection of themare presented here in detail.

Cardiovascular diseases (CVD)(heart disease and stroke)The data for the CVD protective ben-efits of vitamin C are inconsistent.While some studies have failed tofind significant reductions in the riskof coronary heart disease (CHD),numerous prospective cohort stud-ies have found inverse associationsbetween dietary vitamin C intake orvitamin C plasma levels and CVDrisk. Vitamin C may protect coronaryarteries by reducing the build-up of

plaque, as this helps to prevent theoxidation of LDL cholesterol (the“bad” cholesterol), especially incombination with vitamin E. Somedata has shown that vitamin C mayalso boost blood levels of HDL cho-lesterol (the “good” cholesterol),which is also considered positive forthe prevention of heart diseases.The risk of stroke may be reducedby an adequate intake of vitamin Cthrough fruits, vegetables and sup-plements. However, due to theinconsistency of the data and itslack of specificity to vitamin C, theinterpretation of these results is diffi-cult.

CancerThe role of vitamin C in cancer pre-vention has been studied extensive-ly, and until now no beneficial effecthas been shown for breast, prostate,or lung cancer. However, a numberof studies have associated higherintakes of vitamin C with decreasedincidence of cancers of the upperdigestive tract, cervix, ovary, blad-der, and colon. Studies finding sig-nificant cancer risk reduction bydietary intake recommended at least5 servings of fruits and vegetablesper day. Five servings of most fruitsand vegetables provide more than200 mg vitamin C per day. Just sig-nificant cancer risk reductions werefound in people consuming at least80 to 110 mg of vitamin C daily.

Common coldNumerous studies have shown a

general lack of effect ofprophylactic vitaminC supplementationon the incidence ofcommon cold, butthey do show a

moderate benefit interms of the duration

and severity ofepisodes in some

groups, especially thosewho are exposed to sub-

stantial physical and/orcold stress. The improve-

ment in severity of colds after vita-min C supplementation may be dueto the antihistaminic action of megadoses of vitamin C.

Wound healingDuring a postoperative period, orduring healing of superficial wounds,supplemental vitamin C contributesto the prevention of infections andpromotes skin repair.

Blood pressureSeveral studies have shown a bloodpressure lowering effect of vitamin Csupplementation at about 500 mgper day due to improved dilation ofblood vessels.


The recommended daily intake ofvitamin C varies according to age,sex, risk group and criteria appliedin individual countries. The RDAs inthe USA for vitamin C were recentlyrevised upwards to 90 mg/day formen and 75 mg/day for women,based on pharmacokinetic data. Forsmokers, these RDAs are increasedby an additional 35 mg/day. Higheramounts of vitamin C are also rec-ommended for pregnant (85 mg/day)and lactating women (120 mg/day).The RDAs are in a similar range inother countries. Recent evidencesets the estimate for the mainte-nance of optimal health in the regionof 100 mg daily.

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Safety

As much as 6-10 g vitamin C perday (more than 100 times the RDA)has been ingested regularly by manypeople with no evidence of sideeffects. Although a number of possi-ble problems with very large dosesof vitamin C have been suggested,none of these adverse health effectshave been confirmed, and there isno reliable scientific evidence thatlarge amounts of vitamin C (up to 10g/day in adults) are toxic. In the year2000 the US Food and NutritionBoard recommended a tolerableupper intake level (UL) for vitamin Cof 2 g (2,000 mg) daily in order toprevent most adults from experienc-ing osmotic diarrhoea and gastroin-testinal disturbances.


Vitamin C is offered in conventionaltablets, effervescent and chewabletablets, time-release tablets, syrups,powders, granules, capsules, dropsand ampoules, either alone or inmultivitamin-mineral preparations.Buffered vitamin C forms are lessacidic, which can be an advantagein terms of preventing gastric irrita-tion. Vitamin C can also be used inthe form of injections (Rx). A numberof fruit juices, fruit flavour drinks andbreakfast cereals are enriched withvitamin C. On average in Europe,vitamin C supplements providebetween 5.8% and 8.3% of totalvitamin C intake.

Uses in food technology

The food industry uses ascorbic acidas a natural antioxidant. This meansthat ascorbic acid, added to food-stuffs during processing or prior topacking, preserves colour, aromaand nutrient content. This use ofascorbic acid has nothing to do withits vitamin action. In meat process-ing, ascorbic acid makes it possibleto reduce both the amount of addednitrite and the residual nitrite contentin the product. The addition ofascorbic acid to fresh flour improvesits baking qualities, thus saving the4-8 weeks of maturation flour wouldnormally have to undergo aftermilling.


The synthesis of ascorbic acid wasachieved by Reichstein in 1933, andthis was followed by industrial pro-duction five years later by HoffmanLa Roche Ltd. (the vitamin division ofwhich is now DSM NutritionalProducts Ltd.). Today synthetic vita-min C, identical to that occurring innature, is produced from glucose onan industrial scale by chemical andbiotechnological synthesis.


Dietary Reference Intakes*

Infants , 6 months 40mg (Adequate Intake, AI)

Infants 7-12 months 50mg (AI)

Children 1-3 years 15mg



Males 14-18 years 75mg

Females 14-18 years 65mg

Males . 19 years 90mg

Females . 19 years 75mg

Pregnancy , 18 years 80mg

Pregnancy . 19 years 85mg

Lactation , 18 years 115mg

Lactation . 19 years 120mg












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HistoryCa. 400 BC Hippocrates describes the symptoms of scurvy.

1747 British naval physician James Lind prescribes oranges andlemons as a cure for scurvy.

1907 Scurvy is experimentally produced in guinea pigs by Hoist andFrohlich.

1917 A bioassay is developed by Chick and Hume to determine theanti-scorbutic properties of foods.

1930 Szent-Györgyi demonstrates that the hexuronic acid he firstisolated from the adrenal glands of pigs in 1928 is identical tovitamin C, which he extracts in large quantities from sweet pep-pers.

1932 In independent efforts, Haworth and King establish the chemicalstructure of vitamin C.

1932 The relationship between vitamin C and anti-scorbutic factor isdiscovered by Szent-Györgyi and at the same time by King andWaugh.

1933 In Basle, Reichstein synthesises ascorbic acid identical to natural vitamin C. This is the first step towards the vitamin'sindustrial production in 1936.

1937 Haworth and Szent-Györgyi receive a Nobel prize for theirresearch on vitamin C.

1940 In a self experiment, Crandon proves the mandatory contributionof vitamin C in wound healing

1970 Pauling draws worldwide attention with his controversialbestseller “Vitamin C and the Common Cold”.

1975 Experimental studies in vitro illustrate the antioxidant and singlet--79 oxygen quenching properties of vitamin C.

1979 Packer and coworkers observe the free radical interaction ofvitamin E and vitamin C.

1982 Niki demonstrates the regeneration of vitamin E by vitamin C inmodel reactions.

1985 The worldwide requirement for vitamin C is estimated at 30,000-35,000 tons per year.

1988 The National Cancer Institute (USA) recognises the inverserelationship between Vitamin C intake and various forms ofcancer, and issues guidelines to increase vitamin C in the diet.

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1998/99 Three studies show that supplementation with vitamin C candramatically lower lead levels.

2004 A systematic review of thirty studies addressing the effect ofsupplemented vitamin C on the duration of colds revealed thatthere was a consistent benefit, with a reduction in duration of 8%to 14%.

2005 Levine calls for a re-evaluation of vitamin C as cancer therapy,especially intravenous vitamin C

2006 A 5 year Japanese study showed that the risk of contractingthree or more colds in the five-year period was decreased by66% by daily intake of a 500-mg vitamin C supplement.

Albert Szent-Györgyi

Linus Carl Pauling

Charles Glen King

Tadeusz Reichstein

45

Vitamin B1

SynonymsThiamin, thiamine, antiberiberi factor, aneurine, antineuritic factor, nervevitamin.

ChemistryPyrimidine and thiazole moiety linked by a methylene bridge – phosphory-lated forms: thiamin monophosphate (TMP), thiamin diphosphate (TDP),thiamin triphosphate (TTP).

NH2

H3C

CH3

S OH

N

N

N+

CI

Molecular formula of vitamin B1-chloride

Thiamin crystals in polarised light

46

Introduction

Thiamin is a water-soluble B-com-plex vitamin. It was the first B vita-min to be identified and one of thefirst organic compounds to berecognised as a vitamin in the1930s. In fact it was through the dis-covery and naming of thiamin thatthe word 'vitamin', from the Latin“vita” = life and “amine” = nitrogen-containing compound, was coined.The notion that the absence of asubstance in food could cause adisease (in this case beriberi) was arevolutionary one. Man and otherprimates rely on their food intake tocover their vitamin B1 requirements.

Functions

The main functions of thiamin areconnected to its role as a coenzymein the form of thiamin pyrophoshate(TPP). Coenzymes are 'helper mole-cules' which activate enzymes, theproteins that control the thousandsof biochemical processes occurringin the body. TPP acts as a “helpermolecule” in about 25 enzymaticreactions and plays an essential rolein the production of energy fromfood in the carbohydrate metabolismas well as in the links between car-bohydrate, protein and fat metabo-lism. It is one of the key compoundsfor several reactions in the break-down of glucose to energy.Furthermore, TPP is coenzyme forthe metabolism of branched-chainketo acids that are derived frombranched-chain amino acids.

Another important function of thia-min is its activation of an enzymecalled “transketolase”, which in turncatalyses reactions in the pentosephosphate pathway. This pathway isthe basis for the production of manyprominent compounds, such as ATP,GTP, NADPH and the nucleic acidsDNA and RNA.

Certain non-coenzyme functions ofthiamin are important for nerve tis-sues and muscles. Its triphosphateform (TTP) in particular plays a rolein the conduction of nerve impulses,in the metabolism of the neurotrans-mitters acetylcholin, adrenalin, andserotonin and in aerobic metabo-lism.

Main functions in a nutshell:• Co-enzyme in energy

metabolism• Co-enzyme for pentose

metabolism as a basis fornucleic acids

• Nerve impulse conduction andmuscle action

Dietary sources

Thiamin is found in most foods, butmostly in small amounts. The bestsource of thiamin is dried brewer’syeast. Other good sources includemeat (especially pork and ham prod-ucts), some species of fish (eel,tuna), whole grain cereals andbread, nuts, pulses, dried legumesand potatoes. Concerning cerealgrains, the thiamin-rich bran isremoved during the milling of wheatto produce white flour, and duringthe polishing of brown rice to pro-duce white rice. As a consequence,enriched and fortified grain-productsare common today.


Gastrointestinal absorption of nutri-tional thiamin occurs in the lumen ofthe small intestine (mainly thejejunum) by means of a sodium andenergy dependent active transportmechanism. For thiamin levels high-er than 2 µmol/L, passive diffusionplays an additional role. Thiaminoccurs in the human body as freethiamin and its phosphorylated

Food Vitamin B1(mg/100g)

Brewer’s yeast 12

Wheat germ 2

Sunflower seeds 1.5

Brazil nuts 1

Pork 0.9

Beans 0.8

Oatmeal 0.59

Beef 0.23

Vitamin B1 content of foods


forms (see Chemistry). Becausethiamin has a high turnover rate (10-20 days) and is not appreciablystored in the body (approx. 1mg/day is used up in tissues), a dailysupply is required. The limited storesmay be depleted within two weeksor less on a thiamin-free diet, withclinical signs of deficiency beginningshortly after. Regular intake ofvitamin B1 is therefore critical. Theheart, kidney, liver and brain havethe highest concentrations, followedby the leukocytes and red bloodcells.

Measurement

The standard way to assess thiaminstatus used to be to determineerythrocyte transketolase (a-ETK)activity both with and without stimu-lation of this enzyme by the additionof TDP cofactor. Technical difficultiesled to an increasing use of directdetermination of TDP in wholeblood, e.g. by HPLC (High Perfor-mance Liquid Chromatography), inorder to assess thiamin status. TheHPLC assay is more robust andeasier to perform. Thiamin statusdetermined by this method isconsidered to be in good correlationwith results from transketolaseactivation assays. Usually, wholeblood concentrations are found tobe between 66.5 and 200 nmol/L.

Typical serum level <75 nmol/L

Stability

Vitamin B1 is unstable when exposedto heat, alkali, oxygen and radiation.Water solubility is also a factor in the

loss of thiamin from foods. About25% of the thiamin in food islost during the normal cookingprocess. Considerable

amounts may be lost in thaw dripfrom frozen meats or in the water usedto cook meats and vegetables. To pre-serve thiamin, foods should becooked in a covered pan for the short-est time possible and should not besoaked in water or heated for toolong. Juices and cooking water shouldbe re-used in stews and sauces.

Interactions

Positive interactionsThe presence of other B-vitamins,such as vitamins B6, B12, niacin andpantothenic acid, supports theaction of thiamin. Antioxidantvitamins, such as vitamins E and C,protect thiaminby preventing itsoxidation to aninactive form.

Negative interac-tionsA number offoods, such ascoffee, tea, betelnuts (SoutheastAsia) and alsosome cereals mayact as antagoniststo thiamin.Chlorogenic acidand other plantpolyphenols may be responsible forthis anti-thiaminic effect. It is alsoknown that some tropical fish andAfrican silkworms, both traditionallyconsumed raw in some countries,contain enzymes called “thiaminases”that break down vitamin B1. Drugsthat cause nausea and lack of

appetite, or which increase intestinalfunction or urinary excretion, decreasethe availability of thiamin. Poisoningfrom arsenic or other heavy metalsproduces the neurological symptomsof thiamin deficiency. These metals actby blocking a crucial metabolic stepinvolving thiamin in its coenzyme form.

Deficiency

Marginal thiamin deficiency maymanifest itself in such vague symp-toms as fatigue, insomnia, irritabilityand lack of concentration, anorexia,abdominal discomfort, constipationand loss of appetite. When there isnot enough thiamin, the overalldecrease in carbohydrate meta-bolism and its interconnection withamino acid metabolism has severeconsequences. The two principalthiamin deficiency diseases are“beriberi” and “Wernicke-Korsakoffsyndrome”.

Beriberi, which translated intoEnglish means “I can't, I can't”,

manifests itselfprimarily in disor-ders of the nerv-ous and cardio-vascular systems.Unfortunately thisserious disease isstil l common inparts of south-east Asia, wherepolished rice is astaple food andthiamin enrich-ment programsare not fully inplace. Many othercountries fortify

rice and other cereal grains toreplace the nutrients lost in process-ing.

“A certain very troublesomeaffliction, which attacks men,

is called by the inhabitants Beriberi (which means sheep).

I believe those, whom this samedisease attacks, with their kneesshaking and legs raised up, walklike sheep. It is a kind of paraly-sis, or rather Tremor: for it pene-trates the motion and sensation

of the hands and feet indeedsometimes the whole body...”

Jacobus Bonitus, Java, 1630

48

The disease exists in three forms:• dry beriberi, a polyneuropathy with

severe muscle wasting• wet beriberi, which in addition to

neurologic symptoms is charac-terised by cardiovascular manifes-tations, edema and ultimatelyheart failure

• infantile beriberi, which occurs inbreast-fed infants whose nursingmothers are deficient in thiamin.Symptoms of vomiting, convul-sions, abdominal distention andanorexia appear quite suddenlyand may be followed by deathfrom heart failure.

The “Wernicke-Korsakoff syndrome”(cerebral beriberi) is the thiamin defi-ciency disease seen most often inthe Western world. It is frequentlyassociated with chronic alcoholismin conjunction with limited foodconsumption. Symptoms includeconfusion, paralysis of eye motornerves, abnormal oscillation of theeyes, psychosis, confabulation, andimpaired retentive memory andcognitive function. The syndrome isalso seen occasionally in peoplewho fast, have chronic vomiting(hook worm) or have gross malnutri-tion due to e.g., AIDS or stomachcancer. If treatment of amnesicsymptoms is delayed, the memorymay be permanently impaired.Recent evidence suggests thatoxidative stress plays an importantrole in the neurologic pathology ofthiamin deficiency.

The development of vitamin B1deficiency can be caused by:

• Alcoholic disease• Inadequate storage and prepa-

ration of food• Increased demand due to preg-

nancy and lactation, heavyphysical exertion, fever andstress, or adolescent growth

• Inadequate nutrition– high carbohydrate intake

(e.g., milled or polished rice,sweeties)

– regular heavy consumption oftea and coffee (Tannin =antithiamin)

– foods such as raw fish orbetel nuts (thiaminases)

• Certain diseases (dysentery,diarrhea, cancer, nausea/vomit-ing, liver diseases, Infections,malaria, AIDS, hyperthyroidism).

• certain drugs (birth-control pills,neuroleptica, some cancerdrugs)

• Long-term parenteral nutrition(e.g. highly concentrated dex-trose infusions)

Disease prevention andtherapeutic useThiamin is specific in the preventionand treatment of beriberi and othermanifestations of vitamin B1 defi-ciency (e.g. Wernicke-Korsakoff,peripheral neuritis). The dosagerange is from 100 mg daily in milddeficiency states to 200-300 mg insevere cases. Thiamin administra-tion is often beneficial in neuritisaccompanied by excessive alcoholconsumption or pregnancy. Withalcoholic and diabetic polyneu-ropathies, the therapeutic dose ismost often in the range of 10-100mg/daily. When alcoholism has ledto delirium tremens, large doses ofvitamin B1, together with other vita-mins should be given by slow injec-tion. Large doses of thiamin (100-600 mg daily) have been advocatedin the treatment of such diverse con-ditions as lumbago, sciatica, trigem-inal neuritis, facial paralysis andoptic neuritis. However, theresponse to such treatment hasbeen variable.


RDA*

Infants , 6 months 0.2mg (Adequate Intake, AI)

Infants 7-12 months 0.3mg (AI)

Children 1-3 years 0.5mg



Males . 14 years 1.2mg

Females 14-18 years 1.0mg

Females . 19 years 1.1mg

Pregnancy 1.4mg

Lactation 1.4mg












49


Because thiamin facilitates energyutilisation, requirements are tied toenergy intake, which can be verymuch dependent on activity levels.For adults, the RDA is 0.5 mg per1000 kcal, which amounts to arange of 1.0-1.1 mg per day forwomen and 1.2-1.5 mg for men,based on an average caloric intake.An additional 0.4-0.5 mg per day arerecommended during pregnancyand lactation. Children's needs arelower: 0.3-0.4 mg/day (infants) and0.7-1.0 mg/day (children), depend-ing on the age and caloric intake ofthe child.

Safety

Thiamin has been found to be welltolerated in healthy people, even atvery high oral doses (up to 200mg/day). Due to its very broad safety margin for oral administrationand long history of safe use, none ofthe official regulatory authorities hasdefined a safe upper limit for thisvitamin. The only reaction found inhumans is of the hypersensitivitytype. In the vast majority of casesthese have occurred after injectionof thiamin in patients with a history

of allergic reactions. For parenteraladministration, the doses thatproduced these reactions variedfrom 5 to 100 mg, though most ofthem occurred at the higher end ofthis range.


Thiamin is mostly formulated incombination with other B-vitamins(B-complex) or included in multi-vitamin supplements. Fortification ofwhite flour, cereals, pasta, bever-ages and rice began in the UnitedStates during the second World War(1939-1945), with other countriesquickly following suit. Fortification ofstaple foods has virtually eradicatedthe B-vitamin deficiency diseases indeveloped nations.


Chemical synthesis of thiamin is acomplicated process, involvingsome 15-17 different steps.Although commercial production ofthiamin was first accomplished in1937, the production did notdevelop on a broad scale until the1950s, when demand rose sharplybecause of food fortification.

Elmer V. McCollum

Robert R. Williams

Casimir Funk

Christian Eijkman

50

History

7th cent. First classical description of beriberi in a 'General Treatise on theEtiology and Symptoms of Diseases' (author: Ch'ao-Yuan-fangWu Ching).

1882 Takaki, surgeon general, dramatically decreases the incidence ofberiberi in the Japanese navy by improving sailors’ diets.

1897 Dutch medical officers Eijkman and Grijns show that the symp-toms of beriberi can be reproduced in chickens fed on polishedrice, and that these symptoms can be prevented or cured byfeeding them rice bran.

1912 Funk isolates the antiberiberi factor from rice bran extracts andcalls it a 'vitamine' - an amine essential for life. The name findsready acceptance and helps to focus attention on the new con-cept of deficiency diseases.

1915 McCollum and Davis propose water-soluble vitamin B1 asantiberiberi factor

1926 Jansen and Donath isolate antiberiberi factor from rice bran.

1927 The British Medical Research Council proposes vitamin B1 asantiberiberi factor.

1936 Williams, who first began experimenting with vitamin B1 andberiberi in Manila around 1910, identifies and publishes thechemical formula and names it thiamin.

1937 The first commercial production of thiamin is accomplished.

1943 Williams and coworkers, and Foltz and colleagues carry outdietary studies that document widespread thiamin deficiency inthe United States.

1943 Standards of identity for enriched flour are created by the USFood and Nutrition Board, requiring that thiamin, niacin,riboflavin and iron be added to white flour.

51

Vitamin B2

SynonymsRiboflavin, riboflavine, vitamin B2, lactoflavin, ovoflavin

Chemistry7,8-dimethyl-10-(1-D-ribityl)isoalloxazin - different redox states: flavochinon(Flox), flavosemichinon (Fl-H), flavohydrochinon (FlredH2).Coenzyme Form(s): FMN (flavin mononucleotide, riboflavin monophos-phate), FAD (flavin adenine dinucleotide, riboflavin adenosine diphosphate).

NH3CO

OH3C N

N

N

H

CH2

C HHO

C HHO

C HHO

CH2OH

Molecular formula of riboflavin

Vitamin B2 crystals in polarised light

52

Introduction Riboflavin is one of the most widelydistributed water-soluble vitamins.The above synonyms, lactoflavin andovoflavin, as well as the termshepatoflavin, verdoflavin anduroflavin, indicate the source fromwhich the vitamin was originallyisolated, i.e. milk, eggs, liver, plantsand urine. The term “flavin” origi-nates from the latin word “flavus”referring to the yellow colour of thisvitamin. The fluorescent riboflavin isalso part of the vitamin B-complex.In the body, riboflavin occurs prima-rily as an integral component of thecoenzymes flavin mononucleotide(FMN) and flavin adenine dinucleo-tide (FAD).

Functions

Flavin coenzymes are essential forenergy production via the respiratorychain, as they act as catalysts in thetransfer of electrons in numerousessential oxidation-reduction reac-tions (redox reactions). They partici-pate in many metabolic reactions ofcarbohydrates, fats and proteins.Riboflavin coenzymes are alsoessential for the conversion of pyri-doxine (vitamin B6) and folic acidinto their coenzyme forms and forthe transformation of tryptophan toniacin. Vitamin B2 also promotesnormal growth and assists in thesynthesis of steroids, red bloodcells, and glycogen. Furthermore, ithelps to maintain the integrity ofmucous membranes, skin, eyes andthe nervous system, and is involvedin the production of adrenaline bythe adrenal glands. Riboflavin is alsoimportant for the antioxidant statuswithin cell systems, both by itselfand as part of the glutathione reduc-tase and xanthine oxidase system.This defence system may also helpdefend against bacterial infectionsand tumour cells.

Main functions in a nutshell:• Oxidation-reduction reactions• Energy production• Antioxidant functions• Conversion of pyridoxine (vita-

min B6) and folic acid into theiractive coenzyme forms

• Growth and reproduction • Growth of skin, hair, and nails

Dietary sources

Riboflavin is present as an essentialconstituent of all living cells, and istherefore widely distributed.However, there are very few richsources in food. Yeast and liver havethe highest concentrations, but theydo not have much relevance intoday´s human nutrition. The mostimportant and common dietarysources are milk and milk products,lean meat, eggs and green leafyvegetables. Cereal grains, althoughpoor sources of riboflavin, areimportant for those who rely oncereals as their main dietary compo-nent. Fortified cereals and bakery-products supply large amounts.Animal sources of riboflavin are bet-ter absorbed than vegetable

sources. In milk from cows, sheepand goats, at least 90% of theriboflavin is in the free form; in mostother sources, it occurs bound toproteins.


Most dietary riboflavin is consumedas a food protein with FMN and FAD.These are released in the stomachby acidification and absorbed in theupper part of the small intestine byan active, rapid, saturable transportmechanism. The rate of absorptionis proportional to intake and increas-es when riboflavin is ingested alongwith other foods. So approximately15% is absorbed if taken alone ver-sus 60% absorption when takenwith food. Passive diffusion playsonly a minor role at the physiologicaldoses ingested in the diet.In the mucosal cells of the intestine,riboflavin is converted to the coen-zyme form flavin mononucleotide(FMN). In the portal system it isbound to plasma albumin or to otherproteins, mainly immunoglobulins,and transported to the liver, where itis converted to the other coenzymeform, FAD, and bound to specificproteins as flavoproteins.

Riboflavin, mainly as FAD, is distrib-uted in all tissues, but concentra-tions are low and little is stored. Theliver and retinal tissues are the mainstorage places. Riboflavin is excret-ed mainly in the urine where it con-tributes to the yellow colour. Smallamounts are also excreted in sweatand bile. During lactation, about10% of absorbed riboflavin passesinto the milk.


Brewer’s yeast 3.7

Pork liver 3.2

Chicken breast 0.9

Wheat germ 0.7

Camembert/Parmesan 0.6

White mushrooms 0.4

Egg 0.3

Spinach 0.23

Milk/Yoghurt 0.18



53

Measurement

Body status can be determined bydirect and indirect methods. Directmethods include the determinationof FAD and FMN in whole blood byHPLC (High Performance LiquidChromatography). Usually, wholeblood concentrations (FAD) of 175 -475 nmol/L are measured. Anotherpossibil ity for riboflavin statusassessment is the monitoring of uri-nary excretion. Values < 27 µg/gcreatinine point to deficiency, 27 -79 µg/g creatinine are consideredmarginal, and values > 80 µg/g cre-atinine are considered normal.Urinary excretion rises sharply aftertissue saturation is reached. Indirect methods include determin-ing the activity of the FAD dependenterythrocyte glutathion reductase(EGR). This biochemical methodgives a valid indication of riboflavinstatus. During riboflavin deficiencyEGR is no longer saturated withFAD, so enzyme activity increaseswhen FAD is added in vitro. The dif-ference in activity in erythrocytes

with and without added FAD iscalled the activity coefficient(EGRAC). An EGRAC >1.30 isindicative of biochemical riboflavindeficiency.

Stability

Because riboflavin is degraded bylight, loss may be up to 50% if foodsare left out in sunlight or any UVlight. Because of this light sensitivity,riboflavin will rapidly disappear frommilk kept in glass bottles exposed tothe sun or bright daylight (85% with-in 2 hours).Riboflavin is stable when heated andso is not easily destroyed in the ordi-nary processes of cooking, but it willleach into cooking water. The pas-teurisation process causes milk tolose about 20% of its riboflavin con-tent. Alkalis such as baking sodaalso destroy riboflavin. Sterilisationof foods by irradiation or treatmentwith ethylene oxide may also causedestruction of riboflavin.

Interactions

Positive interactionsThyroxine and triiodothyroxine stim-ulate the synthesis of flavin mononu-cleotide (FMN) and flavin adeninedinucleotide (FAD) in mammaliansystems. Anticholinergic drugsincrease the absorption of riboflavinby allowing it to stay longer atabsorption sites.

Negative interactionsCertain drugs have a negative influ-ence on the absorption or metabo-lism of riboflavin:• Ouabain (treatment of congestive

heart failure), theophylline (musclerelaxant, diuretic, central nervousstimulant), and penicillin displaceriboflavin from its binding protein,thus inhibiting transport to thecentral nervous system.

• Probenecid (anti-gout remedy)inhibits gastrointestinal absorptionand renal tubular secretion ofriboflavin.

• Chlorpromazin (anti-psychoticdrug), barbiturates and possiblytricyclic antidepressants preventthe incorporation of riboflavin intoFAD .

• Antibiotics: Riboflavin impairs theantibiotic activity of streptomycin,erythromycin, tyrothricin, car-bomycin and tetracyclines, but noinactivation occurs with chloram-phenicol, penicillin or neomycin.

Deficiency

Overt clinical symptoms of riboflavindeficiency are rarely seen in devel-oped countries. However, the sub-clinical stage of deficiency, charac-terised by a change in biochemicalindices, is common. Riboflavin defi-ciency rarely occurs in isolation butusually in combination with deficien-cies of other B-complex vitamins,because flavoproteins are alsoinvolved in the metabolism of other

54

B-complex vitamins. Along withother B-vitamins, low vitamin B2 sta-tus has been associated withunfavourably increased plasmahomocysteine levels. The absorptionof iron, zinc and calcium is impairedin riboflavin deficiency.

Clinically, vitamin B2-defiency affectsmany organs and tissues. Mostprominent are the effects on theskin, mucosa and eyes:• glossitis (magenta tongue, geo-

graphical tongue)• cheilosis, angular stomatitis (fis-

sures at the corners of the mouth)• sore throat• burning of the lips, mouth, and

tongue• inflamed mucous membranes• pruritus (itching)• seborrheic dermatitis (moist scaly

skin inflammation)• corneal vascularisation associated

with sensitivity to bright l ight,impaired vision, itching and a feel-ing of grittiness in the eyes

In severe long-term deficiency, dam-age to nerve tissue can causedepression and hysteria. Other symptoms are normocytic andnormochromic anaemia, and periph-eral neuropathy of the extremities(tingling, coldness and pain). Lowintracellular levels of flavin coen-zymes could effect mitochondrialfunction, oxidative stress and bloodvessel dilation, which have beenassociated with pre-eclampsia dur-ing pregnancy.

Groups at risk of deficiencyIndividuals who have inadequatefood intake are at risk of deficiency,particularly children from low socio-economic backgrounds in develop-ing countries, elderly people withpoor diets, chronic ‘dieters’, andpeople who exclude milk productsfrom their diet (vegans). Riboflavindeficiency may also occur as a resultof:• trauma, including burns and

surgery• chronic disorders (e.g. rheumatic

fever, tuberculosis, subacutebacterial endocarditis, diabetes,hypothyroidism, liver cirrhosis)

• intestinal malabsorption, e.g.morbus crohn, sprue, lactoseintolerance

• chronic medication (tranquillisers,oral-contraceptives, thyroid hor-mones, fibre-based laxatives,antibiotics)

• high physical activity• phototherapy for newborns during

icterusThe consequences of a lowriboflavin intake may be aggravated

by chronic alcoholism and chronicstress. During pregnancy and lacta-tion riboflavin requirement isincreased.


Eye-related diseasesOxidative damage of lens proteins bylight may lead to the development ofage-related cataracts. Riboflavin defi-ciency leads to decreased glu-tathione reductase activity, which canresult in cataracts. Therefore,riboflavin is used in combination withother antioxidants, like vitamin C andcarotenoids, in disease prevention forage-related cataracts. Riboflavin hasbeen used to treat corneal ulcers,photophobia and noninfective con-junctivitis in patients without any typ-ical signs of deficiency, with benefi-cial results. Most cases of riboflavindeficiency respond to daily oral dosesof 5-10 mg.


RDA*









Pregnancy 1.4mg

Lactation 1.6mg












55

MigrainesPeople suffering from migraineheadaches have a modified mito-chondrial oxygen metabolism.Because riboflavin plays an impor-tant role in energy production,supplemental riboflavin has beeninvestigated as a treatment formigraine. The effect of riboflavinsupplementation at 400 mg /day for3 months was a decrease in gravityand frequency of migraine attacks.

Prevention of deficiencies in high-risk patientsPatients suffering from achlorhydria,vomiting, diarrhoea, hepatic dis-ease, or other disorders preventingabsorption or utilisation, should betreated parenterally. Deficiencysymptoms begin to improve in 1-3days, but complete resolution maytake weeks.


Dietary recommendations forriboflavin exist in many countries,where mean values for adult malesvary between 1.2 and 2.2 mg daily.The recommendations of the Foodand Nutrition Board of the US

National Research Council arebased on feeding studies conductedin the 1940s, which showed that ariboflavin intake of 0.55 mg or lessper day results in clinical signs ofdeficiency after about 90 days.These data have led to the assump-tion that an intake of 0.6 mg per1000 kcal should supply the needsfor essentially all healthy people.

Safety

Riboflavin is extremely nontoxic. Nocases of toxicity from ingestion ofriboflavin have been reported. Notoxic or adverse reactions toriboflavin in humans have been iden-tified. A harmless yellow discol-oration of urine occurs at highdoses. The limited capacity of thegastrointestinal tract to absorb thisvitamin makes any significant riskunlikely, and because riboflavin iswater-soluble, excess amounts aresimply excreted.


Riboflavin is available as oral prepa-rations, alone or most commonly inmultivitamin and vitamin B-complex

preparations, and as an injectablesolution. Crystalline riboflavin (E101)is poorly soluble in water, soriboflavin-5'-phosphate (E 106), amore expensive but more solubleform of riboflavin, has been devel-oped for use in liquid formulations.Riboflavin is one of the vitaminsoften added to flour and bakeryproducts and beverages to compen-sate for losses due to processing. Itis also used to enrich milk, breakfastcereals and dietetic products.Because of its bright yellow colour,riboflavin is sometimes added toother drugs or infusion solutions asa marker.


Riboflavin can be produced bychemical synthesis or by fermenta-tion processes. Chemical processesare usually refinements of the proce-dures developed by Kuhn and byKarrer in 1934 using o-xylene, D-ribose and alloxan as startingmaterials. Various bacteria and fungiare commercially employed tosynthesise riboflavin, using cheapnatural materials and industrialwastes as a growth medium.

Richard Kuhn

Paul Karrer

Otto Heinrich Warburg

56

History

1879 Blyth isolates lactochrome – a water-soluble, yellow fluorescentmaterial – from whey.

1932 Warburg and Christian extract a yellow enzyme from brewer'syeast and suggest that it plays an important part in cell respira-tion.

1933 Kuhn and coworkers obtain a crystalline yellow pigment withgrowth-promoting properties from egg white and whey, whichthey identify as vitamin B2.

1934 Kuhn and associates in Heidelberg, and Karrer and colleagues inZurich synthesise pure riboflavin.

1937 The Council on Pharmacy and Chemistry of the AmericanMedical Association names the vitamin ’riboflavin’.

1937 Theorell determines the structure of flavin mononucleotide, FMN.

1938 Warburg and Christian isolate and characterise flavin adeninedinucleotide (FAD) and demonstrate its involvement as acoenzyme.

1941 Sebrell and coworkers demonstrate clinical signs of riboflavindeficiency in human feeding experiments.

1968 Glatzle and associates propose the use of the erythrocyteglutathione reductase test as a measurement of riboflavin status.

57

Vitamin B6

SynonymsVitamin B6 is composed of three forms (vitamers): pyridoxine or pyridoxol(the alcohol), pyridoxal (the aldehyde) and pyridoxamine (the amine).

ChemistryPyridoxine (3-hydroxy-2-methylpyridine) is a basal compound of the group.Substitution (R) is carried out on 5'-C. Pyridoxic acid is an inactive catabo-lite of the compounds.

H3C

HOR

CH2OH

R = CH2OH = PyridoxineR = CHO = PyridoxalR = CH2NH2 = Pyridoxamine

N

Molecular formulae of vitamin B6

Pyridoxine crystals in polarised light

58

IntroductionPyridoxine is a water-soluble vita-min. Man and other primatesdepend on external sources to covertheir vitamin B6 requirements.Vitamin B6 was discovered in the1930s almost as a by-product of thestudies on pellagra, a deficiency dis-ease caused by the absence in thebody of the vitamin niacin. Negligibleamounts of vitamin B6 can be syn-thesised by intestinal bacteria. Thereare three different natural forms(vitamers) of vitamin B6, namely pyri-doxine, pyridoxamine, and pyridoxal,all of which are normally present infoods. For human metabolism theactive derivative of the vitamin, pyri-doxal 5`-phosphate (PLP), is ofmajor importance as the metaboli-cally active coenzyme form.

Functions

Vitamin B6 serves as a coenzyme ofapproximately 100 enzymes thatcatalyse essential chemical reac-tions in the human body. It plays animportant role in protein, carbohy-drate and lipid metabolism. Its majorfunction is the production of sero-tonin from the amino acid trytophanin the brain and other neurotransmit-ters, and so it has a role in the regu-lation of mental processes andmood. Furthermore, it is involved inthe conversion of tryptophan to thevitamin niacin, the formation ofhaemoglobin and the growth of redblood cells, the absorption of vita-min B12, the production ofprostaglandines and hydrochloricacid in the gastrointestinal tract, thesodium-potassium balance, and inhistamine metabolism. As part of thevitamin B-complex it may also beinvolved in the downregulation of thehomocysteine blood level. VitaminB6 also plays a role in the improve-ment of the immune system.

Main functions in a nutshell:• Nervous system (neurotrans-

mitter synthesis)• Red blood cell formation• Niacin formation• Homocysteine downregulation

(preventing atherosclerosis)• Immune system (antibody

production)• Steroid hormones (inhibition

of the binding of steroidhormones)

Dietary sources

Vitamin B6 is widely distributed infoods, mainly in bound forms.Pyridoxine is found especially inplants, whereas pyridoxal and pyri-doxamine are principally found inanimal tissue, mainly in the form ofPLP. Excellent sources of pyridoxineare chicken and the liver of beef,pork and veal. Good sources includefish (salmon, tuna, sardines, halibut,herring), nuts (walnuts, peanuts),bread, corn and whole grain cereals.Generally, vegetables and fruits arerather poor sources of vitamin B6,although there are products in thesefood classes which contain consid-erable amounts of pyridoxine, suchas lentils, courgettes and bananas.


All three forms of vitamin B6 (pyri-doxine, pyridoxal and pyridoxamine)are readily absorbed in the smallintestine by an energy dependentprocess. All three are converted topyridoxal phosphate in the liver, aprocess which requires zinc andriboflavin. The bioavailabil ity ofplant-based foods varies consider-ably, ranging from 0% to 80%. Someplants contain pyridoxine glycosidesthat cannot be hydrolysed by intes-



Salmon 0.98

Walnuts 0.87

Wheat germ 0.72

Pork liver 0.59

Lentils 0.57

Avocado 0.53

Chicken 0.5

Courgettes 0.46

Bananas 0.36



59

tinal enzymes. Although these glyco-sides may be absorbed, they do notcontribute to vitamin activity.The storage capacity of water-solu-ble vitamins is generally low com-pared to that of fat-soluble ones.Small quantities of pyridoxine arewidely distributed in body tissue,mainly as PLP in the liver and mus-cle. PLP is tightly bound to the pro-teins albumin and haemoglobin inplasma and red blood cells.Because the half-life of pyridoxine is15-20 days and it is not significantlybound to plasma proteins, and thelimited stores may be depleted with-in two to six weeks on a pyridoxin-free diet, a daily supply is required.Excess pyridoxine is excreted in theurine.

Measurement

There are several direct and indirectmethods that can be used forassessing a person’s vitamin B6 sta-tus. Direct methods include determi-nation of PLP in whole blood, and

determination of urinary excretion of4-pyridoxic acid (4-PA). The methodof choice for quantification of bothcompounds is high performanceliquid chromatography. Whole bloodconcentrations usually 35-110nmol/L PLP. Concentrations of PLPhave been found to correlate wellwith the pyridoxine status deter-mined by indirect methods. Indirectmethods measure the stimulatedactivity of pyridoxine dependentenzymes in erythrocytes by additionof PLP. This mainly determines theerythrocyte alanine aminotrans-ferase activation coefficient (EAST-AC) or the erythrocyte aspartateaminotransferase activation coeffi-cient. The coefficient of activity withstimulation to activity without stimu-

lation indicates the pyridoxinestatus. For EAST-AC, values > 1.8are considered to show deficiency,

1.7-1.8 to be marginal,and < 1.7 to be adequate.

For large-scale populationsurveys there is anothermethod of assessing a pyri-

doxine deficiency state:the tryptophan load test.

Vitamin B6 participatesin the conversion oftryptophan to thevitamin niacin. A pyri-doxine deficiency

blocks this process, producing morexanthurenic acid. If the administra-tion of tryptophan leads to anincreased excretion of xanthurenicacid, a pyridoxine deficiency can bediagnosed.Typical serum level of pyridoxine =15-37 nmol/L.

Stability

Pyridoxine is relatively stable toheat, but pyridoxal and pyridoxa-mine are not. Pasteurisation there-fore causes milk to lose up to 20%of its vitamin B6 content. Vitamin B6is decomposed by oxidation andultraviolet light, and by an alkalineenvironment. Because of this lightsensitivity, vitamin B6 will disappear(50% within a few hours) from milkkept in glass bottles exposed to thesun or bright daylight. Alkalis, suchas baking soda, also destroy pyri-doxine. Freezing of vegetables caus-es a reduction of up to 25%, whilemilling of cereals leads to wastes ashigh as 90%. Cooking losses ofprocessed foods may range from afew percent to nearly half the vitaminB6 originally present. Cooking andstorage losses are greater with ani-mal products.

Interactions

Positive interactionsCertain vitamins of the B-complex(niacin, riboflavin, biotin) may actsynergistically with pyridoxine.

Negative interactionsPyridoxine requires riboflavin, zincand magnesium to fulfil its physio-logical function in humans. It hasbeen claimed that women taking oralcontraceptives may have anincreased requirement for pyridox-ine. There are more than 40 drugsthat interfere with vitamin B6, poten-tially causing decreased availabilityand poor vitamin B6 status.Supplementation with the affectednutrient may be necessary. Principalantagonists include:• Phenytoin (an antiepileptic drug)• Theophylline (a drug for respiratory

diseases)• Phenobarbitone (a barbiturate

mainly used for its antiepilepticproperties)

60

• Desoxypyridoxine, an effectiveantimetabolite

• Isoniazid (a tuberculostatic drug)• Hydralazine (an antihypertensive)• Cycloserine (an antibiotic)• Penicillamine (used in treatment of

Wilson’s disease)

Vitamin B6, for its part, reduces thetherapeutic effect of levodopa – anaturally occurring amino acid usedto treat Parkinson’s disease – byaccelerating its metabolism.

Deficiency

A deficiency of vitamin B6 alone isuncommon, because it usuallyoccurs in combination with a deficitin other B-complex vitamins, espe-cially with riboflavin deficiency,because riboflavin is needed for theformation of the coenzyme PLP. Adietary deficiency state showingdefinable clinical deficiency symp-toms is rare, although recent dietsurveys revealed that a significantpart of the following populationgroups have B6 intakes below theRDA:• pregnant and lactating women

(additional demands)• most women in general, especially

those taking oral contraceptives• the elderly (due to lower food

intake)• underweight people• chronic alcoholics (heavy drinking

may severely impair the ability ofthe liver to synthesise PLP, lowintake)

• people with a high protein intake

A pyridoxine depleted diet, anantagonist-induced deficiency orcertain genetic errors of amino acidmetabolism may result in varioussymptoms, such as:• hypochromic anaemia (abnormal

decrease in the haemoglobin con-tent of erythrocytes)

• nervous system dysfunction(decrease in the metabolism of

glutamate in the brain)• impairment of the immune system

(decrease in circulating lympho-cytes)

• epileptiform convulsions in infants• skin lesions, e.g. seborrheic

dermatitis (similar to pellagra)• abdominal distress, nausea,

vomiting• kidney stones• electroencephalographic abnor-

malities• peripheral neuritis, nerve degener-

ation• poor growth• depression, insomnia, lethargy,

decreased alertness• elevated homocysteine

Diseaseprevention andtherapeutic use

Sideroblastic anaemias andpyridoxine-dependent abnormali-ties of metabolismPyridoxine is an approved treatmentfor sideroblastic anaemias and pyri-doxine-dependent abnormalities ofmetabolism. In such cases, thera-peutic doses of approximately 40-200 mg vitamin B6 per day are indi-cated.

PMS (premenstrual syndrome)Some studies suggest that vitaminB6 doses of up to 100 mg/day maybe of value for relieving the symptomcomplex of premenstrual syndrome.However, final conclusions are stilllimited and more research is need-ed.


RDA*






Males 14-50 years 1.3mg





Pregnancy 1.9mg

Lactation 2mg












61

Hyperemesis gravidarumPyridoxine is often administered indoses of up to 40 mg/day in thetreatment of nausea and vomitingduring pregnancy (hyperemesisgravidarum). However, as “morningsickness” improves even withouttreatment it is difficult to prove thetherapeutic benefit.

DepressionPyridoxine is also used to assist inthe relief of depression (especially inwomen taking oral contraceptives).However, clinical trials have not yetprovided evidence for its efficacy.

Carpal tunnel syndromePyridoxine has also been claimed toalleviate the symptoms of carpaltunnel syndrome. Some studiesreport benefits while others do not.

Hyperhomocystinaemia / cardiovas-cular diseaseElevated homocysteine levels in theblood are considered a risk factorfor atherosclerotic disease. Severalstudies have shown that vitamin B6,vitamin B12 and folic acid can lowercritical homocysteine levels.

Immune functionThe elderly are a group that often suf-fers from impaired immune function.Adequate B6 intake is thus important,and it has been shown that theamount of vitamin B6 required toimprove the immune system is higher(2.4 mg/day for men; 1.9 mg/day forwomen) than the current RDA.

AsthmaAsthma patients taking vitamin B6supplements may have fewer andless severe attacks of wheezing,coughing and breathing difficulties.

DiabetesResearch has also suggested thatcertain patients with diabetes melli-tus or gestational diabetes experi-ence an improvement in glucosetolerance when given vitamin B6supplements.

Kidney stonesGlyoxylate can be oxidised to oxalicacid that may lead to calciumoxalate kidney stones. Pyridoxalphosphate is a cofactor for thedegradation of glyoxylate to glycine.There is some evidence that highdoses of vitamin B6 (> 150 mg/day)may be useful for normalising theoxalic acid metabolism to reduce theformation of kidney stones.However, the relationship betweenB6 and kidney stones must bestudied further before any definiteconclusions can be drawn.

Chinese restaurant syndromePeople who are sensitive to gluta-mate, which is often used for thepreparation of Asiatic dishes, canreact with headache, tachycardia(accelerated heart rate), and nausea.50 to 100 mg of pyridoxine can be oftherapeutic value.

AutismHigh dose therapy with pyridoxineimproves the status of autistics inabout 30% of cases.


The recommended daily intake ofvitamin B6 varies according to age,sex, risk group (see ‘Groups at risk’)and criteria applied. The vitamin B6requirement is increased when high-protein diets are consumed, sinceprotein metabolism can only functionproperly with the assistance of pyri-doxine. Pregnant and lactatingwomen need an additional 0.7 mg tocompensate for increased demandsmade by the foetus or baby.

Safety

Vitamin B6 in all its forms is welltolerated, but large excesses aretoxic. Daily oral doses of pyridoxineof up to 50 times the RDA (ca. 100mg) for periods of 3-4 years havebeen administered without adverseeffects. Daily doses of 500 mg andmore may cause sensory neuropathyafter several years of ingestion,whereas the intake of amounts inexcess of 1 gram daily may lead toreversible sensory neuropathy withina few months. Sensory neuropathyhas been selected as a critical end-point on which to base a tolerableupper intake level (UL) of 100mg/day for adults, although supple-ments somewhat higher than thismay be safe for most individuals.Fortunately these side-effects arelargely reversible upon cessation ofvitamin B6 intake. Today, prolongedintake of doses exceeding 500 mg aday is considered to carry the risk ofadverse side-effects.


The most commonly available formof vitamin B6 is pyridoxinehydrochloride, which is used in foodfortification, nutritional supplementsand therapeutic products such ascapsules, tablets and ampoules.Vitamins, mostly of the B-complex,are widely used in the enrichment ofcereals. Dietetic foods such as infantformulas and slimming diets areoften fortified with vitamins, includ-ing pyridoxine.

Paul György

Esmond Emerson Snell

Joseph Goldberger

62

History

1926 Goldberger and coworkers feed rats a diet deficient in what isconsidered to be the pellagra-preventive factor; these animalsdevelop skin lesions.

1934 György first identifies the factor as vitamin B6 or adermin, asubstance capable of curing a characteristic skin disease in rats(dermatitis acrodynia). The factor is then called the rat anti-acro-dynia factor, deficiency of which causes so-called “rat-pellagra”

1935 Birch and György succeed in differentiating riboflavin and vitaminB6 from the specific pellagra preventive factor (P-P) ofGoldberger and his associates.

1938 Lepkovsky is the first to report the isolation of pure crystallinevitamin B6. Independently, but slightly later, several other groupsof researchers also report the isolation of crystallised vitamin B6from rice polishings (Keresztesy and Stevens; György; Kuhn andWendt; Ichiba and Michi).

1939 Harris and Folkers determine the structure of pyridoxine andsucceed in synthesising the vitamin. György proposes the namepyridoxine.

1945 Snell demonstrates that two other natural forms of the vitaminexist, namely pyridoxal and pyridoxamine.

1957 Snyderman determines the levels of vitamin B6 required byhumans.

63

Vitamin B12

SynonymsCobalamin, antipernicious-anaemia factor, Castle’s extrinsic factor, oranimal protein factor.

ChemistryThe structure of vitamin B12 is based on a corrin ring, which has two of thepyrrole rings directly bonded. The central metal ion is Co (cobalt). Four ofthe six coordinations are provided by the corrin ring nitrogens, and a fifth bya dimethylbenzimidazole group. The sixth coordination partner varies, beinga cyano group (-CN) (cyanocobalamin), a hydroxyl group (-OH) (hydroxo-cobalamin), a methyl group (-CH3) (methylcobalamin) or a 5'-deoxyadeno-syl group (5-deoxyadenosylcobalamin).

CH3

CH2CH2CONHCH2C

N

N

CH3

CH3

OCH2OH

HOOPO

O

O

H

NN

H3C

H2NOC–CH2

N NCN

Co+

CH3

H3CH2NOC–CH2–CH2

H2NOC–CH2

CH3

CH3

H

CH2–CONH2

CH2–CH2–CONH2

CH2–CH2–CONH2

CH3

CH3

CH3

Molecular formula of cyanocobalamin

Cyanocobalamin crystals in polarised light

64

Introduction

Vitamin B12 is the largest and mostcomplex of all the vitamins. Thename vitamin B12 is generic for aspecific group of cobalt-containingcorrinoids with biological activity inhumans. Interestingly it is the onlyknown metabolite to contain cobalt,which gives this water-solublevitamin its red colour. This group ofcorrinoids is also known as cobal-amins. The main cobalamins inhumans and animals are hydroxo-cobalamin, adenosylcobalamin andmethylcobalamin, the last two beingthe active coenzyme forms.Cyanocobalamin is a form of vitaminB12 that is widely used clinically dueto its availability and stability. It istransformed into active factors in thebody.In 1934, three researchers won theNobel prize in medicine for discover-ing the lifesaving properties of vita-min B12. They found that eatinglarge amounts of raw liver, whichcontains high amounts of vitaminB12, could save the life of previouslyincurable patients with perniciousanaemia. This finding saves 10,000lives a year in the US alone. VitaminB12 was isolated from liver extract in1948 and its structure was elucida-ted 7 years later.

Functions

Vitamin B12 is necessary for theformation of blood cells, nervesheaths and various proteins. It istherefore, essential for the preven-tion of certain forms of anaemia andneurological disturbances. It is alsoinvolved in fat and carbohydratemetabolism and is essential forgrowth. In humans, vitamin B12functions primarily as a coenzyme inintermediary metabolism. Two meta-bolic reactions are dependent onvitamin B12:

1) The methionine synthase reactionwith methylcobalamin

2) The methylmalonyl CoA mutasereaction with adenosylcobalamin

In its methylcobalamin form vitaminB12 is the direct cofactor for methio-nine synthase, the enzyme that recy-cles homocysteine back to methion-ine. There is evidence that vitaminB12 is required in the synthesis offolate polyglutamates (active coen-zymes required in the formation ofnerve tissue) and in the regenerationof folic acid during red blood cellformation. Methylmalonyl CoA mutase converts1-methylmalonyl CoA to succinylCoA (an important reaction in lipidand carbohydrate metabolism).Adenosylcobalamin is also the coen-zyme in ribonucleotide reduction(which provides building blocks forDNA synthesis).

Main functions in a nutshell:• Essential growth factor• Formation of blood cells and

nerve sheaths• Regeneration of folic acid• Coenzyme-function in the inter-

mediary metabolism, especiallyin cells of the nervous tissue,bone marrow and gastrointesti-nal tract

Dietary sources

Vitamin B12 is produced exclusivelyby microbial synthesis in the diges-tive tract of animals. Therefore, ani-mal protein products are the sourceof vitamin B12 in the human diet, inparticular organ meats (liver, kidney).Other good sources are fish, eggsand dairy products. In foods,hydroxo-, methyl- and 5'-deoxy-adenosyl-cobalamins are the maincobalamins present. Foods of plantorigin contain no vitamin B12 beyondthat derived from microbial contami-nation. Bacteria in the intestinesynthesise vitamin B12, but undernormal circumstances not in areaswhere absorption occurs.

Food Vitamin B12(µg/100g)

Beef liver 65

Crab 27

Blue mussel 8

Steak 5

Coalfish 3.5

Cheese (Camembert) 3

Egg 1-3



65


Vitamin B12 from food sources isbound to proteins and is onlyreleased by an adequate concentra-tion of hydrochloric acid in the stom-ach. Free vitamin B12 is then imme-diately bound to glycoproteins origi-nating from the stomach and salivaryglands. This glycoprotein complexprotects vitamin B12 from chemicaldenaturation. Gastrointestinal ab-sorption of vitamin B12 occurs in thesmall intestine by an active processrequiring the presence of intrinsicfactor, another glycoprotein, whichthe gastric parietal cells secreteafter being stimulated by food. Theabsorption of physiological doses ofvitamin B12 is limited to approxi-mately 10µg/dose. The vitamin B12intrinsic factor complex is thenabsorbed through phagocytosis byspecific i leal receptors. Onceabsorbed, the vitamin is transferredto a plasma-transport protein whichdelivers the vitamin to target cells. Alack of intrinsic factor results in mal-absorption of cobalamin. If this isuntreated, potentially irreversibleneurological damage and life-threat-ening anaemia develops (see defi-ciency).

Regardless of dose, approximately1% of vitamin B12 is absorbed bypassive diffusion, so this processbecomes quantitatively important atpharmacological levels of exposure.Once absorbed, vitamin B12 isstored principally (60%) in the liver.The average B12 content is approxi-mately 1.0 mg in healthy adults, with20-30 µg found in the kidneys,heart, spleen and brain. Estimates oftotal vitamin B12 body content foradults range from 0.6 to 3.9 mg withmean values of 2-3 mg. The normalrange of vitamin B12 plasma concen-trations is 150-750 pg/ml, with peaklevels achieved 8-12 hours afteringestion.

Excretion of vitamin B12 is propor-tional to stores and occurs mainly byurinary and faecal routes. VitaminB12 is very efficiently conserved bythe body, with 65-75% re-absorptionin the ileum of the 0.5-5 µg excretedinto the alimentary tract per day(mainly into the bile). This helps toexplain the slow development (overseveral years) of deficiency states insubjects with negligible vitamin B12intake, such as vegans. Subjectswith a reduced ability to absorbcobalamin via the intestine (lack ofintrinsic factor) develop a deficiencystate more rapidly.

Measurement

Measurement of vitamin B12 in plas-ma is routinely used to determinedeficiency, but may not be a reliableindication in all cases. In pregnancy,for example, tissue levels are normalbut serum levels are low. Vitamin B12can be measured by chemical,microbiological or immunoassay iso-tope dilution methods. Micro-biological assays, which are widelyused for blood and tissue samples,are sensitive but non-specific.Serum cobalamin concentration isoften determined by automatedimmunoassays using intrinsic factoras a binding agent. These assayshave mainly replaced microbiologicalmethods. Data in the literature aboutvitamin B12 concentration in serumvaries. However, values < 110 – 150pmol/L are considered to reflectdeficiency, whereas values > 150 –200 pmol/L represent an adequatestatus.Major vitamin B12-dependent meta-bolic processes include the forma-tion of methionine from homo-cysteine, and the formation of -succinyl coenzyme A from methyl-malonyl coenzyme A. Thus, apartfrom directly determining vitamin B12concentration in the blood, elevatedlevels of both methylmalonic acid(MMA) and homocysteine may

indicate a vitamin B12 deficiency. The Schilling test (which quantifiesileal absorption by measuringradioactivity in the urine after oraladministration of isotopically labelledvitamin) enables detection ofimpaired vitamin B12 absorption. Themeasurement of urinary methyl-malonate (0-3.5 mg/day is normal; avitamin B12-deficient patient willexcrete up to 300 mg/day) can beused as a diagnostic means toassess vitamin B12 status. (Othertests include the cobalaminabsorbance test and the serum gas-trin deoxyuridine suppression test).

Stability

Vitamin B12 is stable to heat, butslowly loses its activity whenexposed to light, oxygen and acid oralkali-containing environments. Lossof activity during cooking is due tothe water solubility of vitamin B12(loss through meat juices or leachinginto water) rather than to its destruc-tion.

Interactions

Negative interactionsAbsorption of cobalamins isimpaired by alcohol and vitamin B6(pyridoxine) deficiency. Furthermore,a number of drugs reduce theabsorption of vitamin B12, and sup-plementation with the affected nutri-ent may be necessary:• Stomach medication: proton pump

inhibitors, H2 receptor antagonists• Liver medication: cholestyramine• Tuberculostatics: para-aminosali-

cylic acid• Anti-gout medication: colchicine• Antibiotics: neomycin, chloram-

phenicol• Anti-diabetics: oral biguanides

metformin and phenformin• Potassium chloride medications• Oral contraceptives

66

A number of anticonvulsants – phe-nobarbitone, primidone, phenytoinand ethylphenacemide – can alterthe metabolism of cobalamins in thecerebrospinal fluid and lead to neu-ropsychic disturbances. Severalsubstituted amide, lactone and lac-tam analogues of cyanocobalamincompete with binding sites on intrin-sic factor and lead to depressedabsorption of the vitamin. Nitrousoxide (anaesthetic) also interfereswith cobalamin metabolism.

Deficiency

Clinical cobalamin deficiency due todietary insufficiency is rare inyounger people, but occurs morefrequently in older people. VitaminB12 deficiency affects 10-15% ofindividuals over the age of 60.

Deficiency of vitamin B12 leads todefective DNA synthesis in cells,which affects the growth and repairof all cells. Tissues most affected arethose with the greatest rate of cellturnover, e.g. those of thehaematopoietic system. This canlead to megaloblastic anaemia(characterised by large and imma-ture red blood cells) and neuropathy,with numerous symptoms including:glossitis, weakness, loss of appetite,loss of taste and smell, impotence,irritability, memory impairment, milddepression, hallucination, breath-lessness (dyspnea) on exertion, tin-gling and numbness (paraesthesia).Vitamin B12 deficiency can also leadto hyperhomocysteinaemia, a possi-ble risk factor for occlusive vasculardisease.

The symptoms of vitamin B12 defi-ciency are similar to those of folicacid deficiency, the major differencebeing only that vitamin B12 deficien-cy is associated with spinal corddegeneration. If folic acid is used totreat vitamin B12 deficiency, anaemiamay be alleviated but the risk of

damage to the nervous systemremains. It is therefore essential todiagnose the deficiency accuratelybefore starting therapy. The Foodand Nutrition Board advises adultsto limit their folic acid intake(through supplements and fortifica-tion) to 1 mg per day.

Cause of deficiency is not usuallyinsufficient dietary intake but lack ofintrinsic factor secretion. Withoutintrinsic factor, absorption is notpossible and a severe and persistentdeficiency develops that cannot beprevented by the usual dietaryintakes of vitamin B12. This occurs inpeople with:

• pernicious anaemia (a hereditaryautoimmune disease that chieflyaffects persons post middle age),

• food-bound vitamin B12 malabsorp-tion, reported in patients on long-term treatment with certain drugs,and in elderly patients with gastricatrophy.

• after gastrectomy• after ingestion of corrosive agents

with destruction of gastric mucosa.• lesions of the small bowel (blind

loops, stenoses, strictures, diver-ticula). Bacterial overgrowth maylead to competitive utilisation ofavailable vitamin. Impaired absorp-tion also occurs in patients withsmall intestinal defects (e.g. sprue,celiac disease, ileitis, ileal resec-tion) and those with inborn errors ofcobalamin metabolism, secretion ofbiologically abnormal intrinsic fac-tor, or Zollinger-Ellison syndrome.

• pancreatic insufficiency• in alcoholics vitamin B12 intake

and absorption are reduced, whileelimination is increased

• AIDS also brings an increased riskof deficiency

The risk of nutritional deficiency isincreased in vegans; a high intake offibre has been shown to aggravate aprecarious vitamin balance. Therehave also been reports of vitaminB12 deficiency in infants breast-fed

by vegetarian mothers. Strict vege-tarians are urged to use a vitaminB12 supplement.

Pernicious anaemia:Pernicious anaemia is the classi-cal symptom of B12 deficiency,but it is actually the end-stage ofan autoimmune inflammation ofthe stomach, resulting in destruc-tion of stomach cells by thebody’s own antibodies. Anaemiais a condition in which red bloodcells do not provide adequateoxygen to body tissues.Pernicious anaemia is a type ofmegaloblastic anaemia.

Gastric atrophy:Gastric atrophy is a chronicinflammation of the stomachresulting in decreased stomachacid production. Because this isnecessary for the release of vita-min B12 from the proteins in food,vitamin B12 absorption is reduced.


Pernicious anaemiaPatients with lack of intrinsic factorsecretion can be effectively treatedusing oral vitamin B12 but requirelifetime vitamin B12 therapy. Whenused alone, oral doses of at least150 µg/day are necessary, althoughsingle weekly oral doses of 1000 µghave proved satisfactory in somecases. Combinations of vitamin B12and intrinsic factor may be given,but as a variable number of patientsbecome refractory to intrinsic factorafter prolonged treatment, parenter-al therapy with cyanocobalamin orhydroxocobalamin is preferred.

67

HyperhomocysteinaemiaHomocysteine appears to be a nerveand vessel toxin, promoting mortali-ty and cardiovascular disease (CVD)as well as stroke, Alzheimer's dis-ease, birth defects, recurrent preg-nancy loss, and eye disorders.Keeping homocysteine at levelsassociated with lower rates ofdisease requires adequate B12, folicacid and B6 intake.

CancerVitamin B12 deficiency may lead toan elevated rate of DNA damage andaltered methylation of DNA. Theseare obvious risk factors for cancer.In a recent study, chromosomebreakage was minimised in youngadults by supplementation with700 µg of folic acid and 7 µg ofvitamin B12 daily in cereal for twomonths.


RDA intakes for vitamin B12 rangefrom 0.3 to 5.0 µg/day in 25 coun-tries. An increase to 2.2 µg/day isrecommended during pregnancyand to 2.6 µg/day for lactation tocover the additional requirements ofthe foetus/infant. The Committee onNutrition of the American Academyof Paediatrics recommends a dailyvitamin B12 intake of 0.15 µg/100kcal energy intake for infants andpreadolescent children. Otherauthorities have suggested intakesof 0.3-0.5 µg (0-1 year of age), 0.7-1.5 µg (1-10 years of age) and 2 µg(> 10 years). The “average” westerndiet probably supplies 3-15 µg/day,but can range from 1-100 µg/day.

Safety

Large intakes of vitamin B12 fromfood or supplements have causedno toxicity in healthy people. Noadverse effects have been reportedfrom single oral doses as high as100 mg and chronic administrationof 1 mg (500 times the RDA) weeklyfor up to 5 years. Moreover, therehave been no reports of carcino-genic or mutagenic properties, andstudies to date indicate no terato-genic potential. The main foodsafety authorities have not set atolerable upper intake level (UL) forvitamin B12 because of its lowtoxicity.


The principal form of vitamin B12used in supplements is cyanocobal-amin. It is available in the form ofinjections and as a nasal gel for thetreatment of pernicious anaemia.Cyanocobalamin is also available intablet and oral liquid form for vitaminB-complex, multivitamin and vitaminB12 supplements.Vitamin B12 is widely used to enrichcereal products and certain bever-ages. Dietetic foods such as slim-ming foods and infant formulas areoften fortified with vitamins, includ-ing vitamin B12. Fortification withvitamin B12 is especially importantfor products aimed at people with alow dietary intake, such as vegans.


Vitamin B12 is produced commer-cially from bacterial fermentation,usually as cyanocobalamin.


RDA*

Infants , 6 months 0.4 µg (Adequate Intake, AI)

Infants 7-12 months 0.5 µg (AI)

Children 1-3 years 0.9 µg



Adults . 14 years 2.4 µg

Pregnancy 2.6 µg

Lactation 2.8 µg












Dorothy Crowfoot Hodgkin

Robert Burns Woodward

William Parry Murphy

68

History

1824 The first case of pernicious anaemia and its possible relation todisorders of the digestive system is described by Combe.

1855 Combe and Addison identify clinical symptoms of perniciousanaemia.

1925 Whipple and Robscheit-Robbins discover the benefit of liver inthe regeneration of blood in anaemic dogs.

1926 Minot and Murphy report that a diet of large quantities of rawliver to patients with pernicious anaemia restores the normal levelof red blood cells. Liver concentrates are developed and studieson the presumed active principle(s) (“antipernicious anaemiafactor”) are initiated.

1929 Castle postulates that two factors are involved in the control ofpernicious anaemia: an “extrinisic factor” in food and an “intrinsic factor” in normal gastric secretion. Simultaneousadministration of these factors causes red blood cell formationwhich alleviates pernicious anaemia.

1934 Whipple, Minot and Murphy are awarded the Nobel prize formedicine for their work in the treatment of pernicious anaemia.

1948 Rickes and associates (USA) and Smith and Parker (England),working separately, isolate a crystalline red pigment which theyname vitamin B12.

1948 West shows that injections of vitamin B12 dramatically benefitpatients with pernicious anaemia.

1949 Pierce and coworkers isolate two crystalline forms of vitamin B12equally effective in combating pernicious anaemia. One form isfound to contain cyanide (cyanocobalamin) while the other is not(hydroxocobalamin).

1955 Hodgkin and coworkers establish the molecular structure ofcyanocobalamin and its coenzyme forms using X-ray crystallog-raphy.

1955 Eschenmoser and colleagues in Switzerland and Woodward andcoworkers in the USA synthesise vitamin B12 from cultures ofcertain bacteria/fungi.

1973 Total chemical synthesis of vitamin B12 by Woodward andcoworkers.

Georg Richard Minot

69

Niacin: Nicotinic Acid and Nicotinamide

SynonymsVitamin B3, vitamin B4, PP factor (pellagra-preventative factor)

ChemistryNicotinic acid (pyridine-3-carboxylic acid), nicotinamide (pyridine-3 carbox-amide)

COOH

N

C – NH2

N

O

Nicotinic Acid Nicotinic Amide

Molecular formula of nicotinic acid

Nicotinic acid crystals in polarised light

70

IntroductionThe term niacin refers to both nico-tinic acid and its amide derivative,nicotinamide (niacinamide). Both areused to form the coenzymes nicoti-namide adenine dinucleotide (NAD)and nicotinamide adenine dinu-cleotide phosphate (NADP). Niacin isa member of the water soluble B- vitamin complex. The amino acidtryptophan can be converted tonicotinic acid in humans, thereforeniacin is not really a vitamin providedthat an adequate dietary supply oftryptophan is available.Nicotinic acid was isolated as earlyas 1867. In 1937 it was demonstra-ted that this substance cures thedisease pellagra. The name niacin isderived from nicotinic acid + vitamin.

Functions

The coenzymes NAD and NADP arerequired for many biological oxida-tion-reduction (redox) reactions.About 200 enzymes require NAD orNADP. NAD is mainly involved inreactions that generate energy in tis-sues by the biochemical degradationof carbohydrates, fats and proteins.NADP functions in reductive biosyn-theses such as the synthesis of fattyacids and cholesterol. NAD is also required as a substratefor non-redox reactions. It is thesource of adenosine diphosphate(ADP)-ribose, which is transferred toproteins by different enzymes. Theseenzymes and their products seem tobe involved in DNA replication, DNArepair, cell differentiation and cellularsignal transduction.

Main functions in a nutshell:• Coenzymes (NAD and NADP) in

redox reactions• NAD is a substrate for non-

redox reactions

Dietary sourcesNicotinamide and nicotinic acidoccur widely in nature. Nicotinic acidis more prevalent in plants, whereasin animals nicotinamide predomi-nates. Yeast, l iver, poultry, leanmeats, nuts and legumes contributemost of the niacin obtained fromfood. Milk and green leafy vegeta-bles contribute lesser amounts.In cereal products (corn, wheat),nicotinic acid is bound to certaincomponents of the cereal and isthus not bioavailable. Specific foodprocessing, such as the treatment ofcorn with lime water involved in thetraditional preparation of tortillas inMexico and Central America,increases the bioavailability of nico-tinic acid in these products.Tryptophan contributes as much astwo thirds of the niacin activityrequired by adults in typical diets.Important food sources of trypto-phan are meat, milk and eggs.


Both acid and amide forms of thevitamin are readily absorbed fromthe stomach and the small intestine.At low concentrations the two formsare absorbed by a sodium-depend-ent facil itated diffusion, and athigher concentrations by passivediffusion. Niacin is present in the dietmainly as NAD and NADP, andnicotinamide is released from thecoenzyme forms by enzymes in theintestine. The main storage organ,the liver, may contain a significantamount of the vitamin, which isstored as NAD. The niacin coen-zymes NAD and NADP are synthe-sised in all tissues from nicotinicacid or nicotinamide.

Measurement

Determination of the urinaryexcretion of two niacin metabo-lites, N-methyl-nicotinamide and N-methyl-2-pyridone-5-carboxam-ide has been used to assess niacinstatus. Excretion of 5.8 ± 3.6 mg N-methyl-nicotinamide/24hrs and20.0 ± 12.9 mg N-methyl-2-pyri-done-5-carboxamide/24hrs areconsidered normal.Recent studies suggest that themeasurement of NAD and NADPconcentrations and their ratio in redblood cells may be sensitive andreliable indicators for the determina-tion of niacin status. A ratio oferythrocyte NAD to NADP < 1.0 mayidentify subjects at risk of develop-ing niacin deficiency.

Food Niacin (mg/100g)

Veal liver 15

Chicken 11

Beef 7.5

Salmon 7.5

Almonds 4.2

Peas 2.4

Potatoes 1.2

Peach 0.9

Tomatoes 0.5

Milk (whole) 0.1

Niacin content of foods


71

Stability

Both nicotinamide and nicotinic acidare stable when exposed to heat,light, air and alkali. Little loss occursin the cooking and storage of foods.

Interactions

Negative interactionsCopper deficiency can inhibit theconversion of tryptophan to niacin.The drug penicillamine has beendemonstrated to inhibit the trypto-phan-to-niacin pathway in humans;this may be due in part to the cop-per-chelating effect of penicillamine. The pathway from tryptophan toniacin is sensitive to a variety ofnutritional alterations. Inadequateiron, riboflavin, or vitamin B6 statusreduces the synthesis of niacin fromtryptophan.Long-term treatment of tuberculosiswith isoniazid may cause niacin defi-ciency because isoniazid is a niacinantagonist. Other drugs which inter-act with niacin metabolism may alsolead to niacin deficiency, e.g. tran-quillisers (diazepam) and anticonvul-sants (phenytoin, phenobarbitol).

Deficiency

Symptoms of a marginal niacin defi-ciency include: insomnia, loss ofappetite, weight and strength loss,soreness of the tongue and mouth,indigestion, abdominal pain, burningsensations in various parts of thebody, vertigo, headaches, numb-ness, nervousness, poor concentra-tion, apprehension, confusion andforgetfulness.

Severe niacin deficiency leads topellagra, a disease characterised bydermatitis, diarrhoea and dementia.In the skin, a pigmented rash devel-ops symmetrically in areas exposed

to sunlight (theterm pellagracomes from theItalian phrase for rawskin). Symptoms affect-ing the digestive system includea bright red tongue, stomatitis,vomiting, and diarrhoea.Headaches, fatigue, depression,apathy, and loss of memory areneurological symptoms of pellagra.If untreated, pellagra is fatal.Since the synthesis of NAD fromtryptophan requires an adequatesupply of riboflavin and vitamin B6,insufficiencies of these vitamins mayalso contribute to niacin deficiency,resulting in pellagra. Pellagra is rarely seen in industri-alised countries, except for itsoccurrence in people with chronicalcoholism. In other parts of theworld where maize and jowar (bar-ley) are the major staples, pellagrapersists. It also occurs in India andparts of China and Africa.Patients with Hartnup’s disease, agenetic disorder, develop pellagrabecause their absorption of trypto-phan is defective. Carcinoid syn-drome may also result in pellagrabecause dietary tryptophan is pref-erentially used for serotonin synthe-sis and NAD synthesis is thereforerestricted.


Niacin is specific in the treatment ofglossitis, dermatitis and the mentalsymptoms seen in pellagra.High doses of nicotinic acid (1.5-4g/day) can reduce total and low-densitiy lipoprotein cholesterol andtriacylglycerols and increase high-density l ipoprotein cholesterol inpatients at risk of cardiovascular dis-ease. There is a flush reaction tohigh doses of nicotinic acid, which is

seen primarily with a rising bloodlevel and may wear off once aplateau level has been reached.Nicotinic acid has also been used indoses of 100 mg as a vasodilator inpatients suffering from diseasescausing vasoconstriction.Type 1 diabetes mellitus results fromthe autoimmune destruction ofinsulin-secreting b-cells in the pan-creas. There is evidence that nicoti-namide may delay or prevent thedevelopment of diabetes. Clinicaltrials are in progress to investigatethis effect of nicotinamide.Recent studies suggest that infec-tion with human immunodeficiencyvirus (HIV) increases the risk ofniacin deficiency. Higher intakes ofniacin were associated withdecreased progression rate to AIDSin an observational study of HIV-positive men.DNA damage is an important riskfactor for cancer. NAD is consumedas a substrate in ADP-ribose trans-fer reactions to proteins which play arole in DNA repair. This has arousedinterest in the relationship betweenniacin and cancer. A large case-con-trol study found increased consump-tion of niacin, along with antioxidantnutrients, to be associated withdecreased incidence of cancers ofthe mouth, throat and oesophagus.


The actual daily requirement ofniacin depends on the quantity oftryptophan in the diet and the effi-ciency of the tryptophan to niacinconversion. The conversion factor is60 mg of tryptophan to 1 mg ofniacin, which is referred to as 1niacin equivalent (NE). This conver-sion factor is used for calculatingboth dietary contributions from tryp-tophan and recommendedallowances of niacin.In the USA, the RDA for adults is 16mg NEs for men and 14 mg NEs forwomen. Other regulatory authoritieshave established similar RDAs.

Safety

There is no evidence that niacin fromfoods causes adverse effects.Pharmacological doses of nicotinicacid, but not nicotinamide, exceed-ing 300 mg per day have been asso-ciated with a variety of side effects

including nausea, diarrhoea andtransient flushing of the skin. Dosesexceeding 2.5 g per day have beenassociated with hepatotoxicity, glu-cose intolerance, hyperglycaemia,elevated blood uric acid levels,heartburn, nausea, headaches.Severe jaundice may occur, evenwith doses as low as 750 mg perday, and may eventually lead to irre-versible liver damage. Doses of 1.5to 5 g/day of nicotinic acid havebeen associated with blurred visionand other eye problems.Tablets with a buffer and timerelease capsules are available toreduce flushing and gastrointestinalirritation for persons with a sensitivi-ty to nicotinic acid. These should beused with caution, however,because time-release niacin tabletsused at high levels are linked to liverdamage.The Food and Nutrition Board (1998)set the tolerable upper intake level(UL) for niacin (nicotinic acid plusnicotinamide) at 35 mg/day. The EUScientific Committee on Food (2002)developed different upper levels fornicotinic acid and nicotinamide: theUL for nicotinic acid has been set at10 mg/day, for nicotinamide at 900mg/day.


Single supplements of nicotinic acidare available in tablets, capsules andsyrups. Multivitamin and B-complexvitamin infusions, tablets and cap-sules also contain nicotinamide.Niacin is used to fortify grain includ-ing corn and bran breakfast cerealsand wheat flour (whole meal, whiteand brown). US standards of identityand state standards require enrich-ment of bread, flour, farina, maca-roni, spaghetti and noodle products,corn meal, corn grits and rice.


Although other routes are known,most nicotinic acid is produced byoxidation of 5-ethyl-2-methylpyri-dine. Nicotinamide is produced via3-methylpyridine. This compound isderived from two carbon sources,acetaldehyde and formaldehyde, orfrom acrolein plus ammonia. 3-Methylpyridine is first oxidised to3-cyanopyridine, which in a secondstage converts to nicotinamide byhydrolysis.Current recommendations in the USA

RDA*

Infants , 6 months 2mg (AI)

Infants 7-12 months 4mg (AI)




Males . 14 years 16mg

Females . 14 years 14mg

Pregnancy 18mg

Lactation 17mg












72

Conrad Elvehjem

Tom Spies

Casimir Funk

73

History

1755 The disease pellagra is first described by Thiery who calls thedisease “mal de la rosa”.

1867 Huber provides the first description of nicotinic acid.

1873 Weidel describes the elemental analysis and crystalline structureof the salts and other derivatives of nicotinic acid in some detail.

1894 First preparation of nicotinamide by Engler.

1913 Funk isolates nicotinic acid from yeast.

1915 Goldberger demonstrates that pellagra is a dietary deficiencydisease.

1928 Goldberger and Wheeler use the experimental model of blacktongue disease in dogs as an experimental model for the humandisease pellagra.

1937 Elvehjem and coworkers show the effectiveness of nicotinic acidand nicotinamide in curing canine black tongue.

1937 Spies cures human pellagra using nicotinamide.

1945 Krehl discovers that the essential amino acid tryptophan is trans-formed into niacin by mammalian tissues.

1955 The concept of niacin equivalents is proposed by Horwitt.

1955 Altschul and associates report that high doses of nicotinic acidreduce serum cholesterol in man.

1961 Turner and Hughes demonstrate that the main absorbed form ofniacin is the amide.

1979 Shepperd and colleagues report that high doses of nicotinic acidlower both serum cholesterol and triglycerides.

1980 Bredehorst and colleagues show that niacin status affects theextent of ADP-ribosylation of proteins.

74

Pantothenic Acid

SynonymsVitamin B5, antidermatosis vitamin, chick antidermatitis factor, chickantipellagra factor

ChemistryPantothenic acid is composed of beta-alanine and 2,4-dihydroxy-3,3-dimethyIbutyric acid (pantoic acid), acid amide-linked. Pantetheine consistsof pantothenic acid linked to a ß-mercaptoethylamine group.

HO CH2NC

CH3

CH3

CH2 C

H

OH

C

O H

CH2 COOH

Molecular formula of pantothenic acid

Calcium pantothenate crystals in polarised light

75

Introduction

Pantothenic acid was discovered in1933 and belongs to the group ofwater-soluble B vitamins. Its nameoriginates from the Greek word“pantos”, meaning “everywhere”, asit can be found throughout all livingcells.

Functions

Pantothenic acid, as a constituent ofcoenzyme A (a coenzyme of acetyla-tion), plays a key role in the metabo-lism of carbohydrates, proteins andfats, and is therefore important forthe maintenance and repair of all cellsand tissues. Coenzyme A is involvedin reactions that supply energy, in thesynthesis of essential lipids (e.g.sphingolipids, phospholipids), sterols(e.g. cholesterol), hormones (e.g.growth, stress and sex hormones),neurotransmitters (e.g. acetylcholine),porphyrin (a component of haemo-globin, the oxygen-carrying red bloodcell pigment) and antibodies, and inthe metabolism of drugs (e.g.sulphonamides) and in alcohol detox-ification. Another essential role ofpantothenic acid concerns acyl carri-er protein, an enzyme involved in thesynthesis of fatty acids. In theprocess of fat burning, pantothenicacid works in concert with coenyzmeQ10 and L-carnitine.

Main functions in a nutshell:• Metabolism of carbohydrates,

proteins and fats• Supply of energy from foods• Synthesis of essential l ipids,

sterols, hormones, neurotrans-mitters, and porphyrin

• Metabolism of drugs andalcohol detoxification

Dietary sources

The active vitamin is present in vir-tually all plant, animal and microbialcells. Thus pantothenic acid is widelydistributed in foods, mostly incorpo-rated into coenzyme A. Its richestsources are yeast and organ meats(liver, kidney, heart, brain), but eggs,milk, vegetables, legumes and whole-grain cereals are more commonsources.

Pantothenic acid is synthesised byintestinal micro-organisms, but theextent and significance of this enteralsynthesis is unknown.


Most of the pantothenic acid in foodexists in the form of coenzyme A, andpantothenic acid is released by aseries of enzyme reactions in thesmall intestine. It is then absorbed bypassive diffusion into the portal circu-lation and transported to the tissues,where re-synthesis of the coenzymeoccurs. About half of the pantothenicacid in the diet is actually absorbed.If calcium pantothenate or pan-

tothenic acid are ingested as nutri-tional supplements, they must first beconverted to pantetheine by intestin-al enzymes before being absorbed.Topical and orally applied D-pan-thenol (the alcoholic form of pan-tothenic acid that can, e.g., be foundin many cosmetic products) is alsoabsorbed by passive diffusion andtransformed to pantothenic acid byenzymatic oxidation. The highestconcentrations in the body are in theliver, adrenal glands, kidneys, brain,heart and testes. Total pantothenicacid levels in whole blood are at least1 mg/L in healthy adults; most of itexists as coenzyme in the red bloodcells. Urinary excretion in the form ofpantothenic acid generally correlateswith dietary intake, but variation islarge (2-7 mg daily). During lactation,a large proportion of the intakereaches the milk (1-5 mg daily).

Measurement

Due to the fact that dietary deficiencyis practically unknown, little researchhas been conducted through assaysto assess pantothenate status inman. Nutritional status can bededuced from amounts of pantothen-ate excreted in urine. Less than 1 mgdaily is considered abnormally low. Amore convenient approach is deter-mination of pantothenate in serum, orpreferably whole blood, by microbio-logical methods. Although theseassays are highly sensitive andspecific, they are slow and tedious toperform. New methods, such asHPLC/MS (High Performance LiquidChromatography / mass spectrome-try) and immunologic methods, havealso been applied. Another methodsuggested for assessing nutritionalstatus is the sulphanilamide acetyla-tion test, which measures the activityof coenzyme A in the blood. Wholeblood levels typically range from 0.9 –1.5 µmol/L.

Food Pantothenic acid (mg/100g)

Veal liver 7.9


Peanuts 2.1

White mushrooms 2.1

Egg 1.6

Wheat germ 1

Herring 0.94

Milk 0.35

Vegetables 0.2-0.6

Pantothenic acid content of foods


76

Stability

Pantothenic acid is stable under neu-tral conditions, but is readilydestroyed by heat in alkaline or acidsolutions. Up to 50% may be lostduring cooking (due to leaching) andup to 80% as a result of food pro-cessing and refining (canning, freez-ing, milling etc.). Pasteurisation ofmilk only causes minor losses.

Interactions

Positive interactionsVarious studies have indicated thatvitamin B12 may aid in the conver-sion of free pantothenic acid intocoenzyme A. In the absence of B12,coenzyme A production isdecreased and fat metabolismimpaired. In animal experiments,ascorbic acid (vitamin C) was shownto lessen the severity of symptomsdue to pantothenic acid deficiency;vitamin A, vitamin B6, folic acid andbiotin are also necessary for properutilisation of pantothenic acid.

Negative interactionsEthanol causes a decrease in theamount of pantothenic acid in tis-sues, with a resulting increase inserum levels. It has therefore beensuggested that pantothenic acid utili-sation is impaired in alcoholics. Birthcontrol pills containing estrogen andprogestin may increase the require-ment for pantothenic acid. The mostcommon antagonist of pantothenicacid used experimentally to acceler-ate the appearance of deficiencysymptoms is omega-methyl pan-tothenic acid. L-pantothenic acid hasalso been shown to have an antago-nistic effect in animal studies. Methylbromide, a fumigant used to controlvermin in places where food isstored, destroys the pantothenic acidin foods exposed to it.

Deficiency

Since pantothenic acid occurs tosome extent in all foods, it is general-ly assumed that dietary deficiency ofthis vitamin is extremely rare.However, pantothenic acid deficiencyin humans is not well documentedand probably does not occur in isola-tion but in conjunction with deficien-cies of other B vitamins. Clinical man-ifestations that can be clearlyascribed to dietary deficiency of pan-tothenic acid have not been identi-fied, although it has been implicatedin “burning feet” syndrome, a condi-tion observed among malnourishedprisoners of war in the 1940s.Deficiency symptoms have been pro-duced experimentally by administer-ing the antagonist omega-methylpantothenic acid. They includefatigue, headaches, insomnia, nau-sea, abdominal cramps, vomiting andflatulence. The subjects complainedof tingling sensations in the arms andlegs, muscle cramps and impairedcoordination. There was cardiovascu-lar instability and impaired responsesto insulin, histamine and ACTH (astress hormone).

Homopantothenate is a pantothenicacid antagonist that has been used inJapan to enhance mental function,especially in Alzheimer's disease. Arare side effect was an abnormalbrain function resulting from the fail-ure of the liver to eliminate toxins(hepatic encephalopathy). This condi-tion was reversed by pantothenicacid supplementation, suggesting itwas due to pantothenic acid deficien-cy caused by the antagonist. Inexperiments with mice it has beenshown that a deficiency of pan-tothenic acid leads to skin irritationand greying of the fur, which werereversed by giving pantothenic acid.Pantothenic acid has since beenadded to shampoo, although it hasnever been successful in restoringhair colour in humans.

Groups at risk of deficiency• Alcoholics• Women on oral contraceptives• People with insufficient food intake

(e.g. elderly, post-operative)• People with impaired absorption

(due to certain internistic diseases)


RDA*

Infants < 6 months 1.7mg (AI)


Children 1-3 years 2mg (AI)



Adults >14 years 5mg (AI)

Pregnancy 6mg (AI)

Lactation 7mg (AI)












77


Although isolated deficiency states arerarely observed, various investigatorshave noted changes in pantothenicacid levels in various diseases, andpharmacological amounts of the vita-min are used in the treatment ofnumerous conditions. In most cases,however, the claimed therapeuticresponses have not been confirmedby controlled studies in humans. For the treatment of deficiency due toimpaired absorption, intravenous orintramuscular injections of 500 mgseveral times a week are recommend-ed. Postoperative ileus (paralysis ofthe intestine) requires doses of up to1000 mg every six hours. Panthenol is applied topically to skinand mucosa to speed healing ofwounds, ulcers and inflammation,such as cuts and grazes, burns, sun-burn, nappy rash, bed sores, laryngitisand bronchitis. In combination, pan-tothenic acid and ascorbic acid signif-icantly enhance post surgical therapyand wound healing. The healingprocess of conjunctiva and the corneaafter reconstructive surgery of theepithelium has also been accelerated.Pantothenic acid has been tried, withvarying results, to treat various liverconditions, arthritis, and constipationin the elderly; to prevent urinary reten-tion after surgery or childbirth; and(together with biotin) to prevent bald-ness. It has also been reported tohave a protective effect against radia-tion sickness.Pantethine is used to normalise lipidprofiles, as it lowers elevated triglyc-erides and LDL cholesterol while rais-ing levels of the beneficial HDL choles-terol. Pantethine actually consists oftwo molecules of pantetheine joinedby two molecules of sulphur (a disul-phide bridge). It is especially effectiveat lowering elevated blood lipids inpatients with diabetes without hinder-ing blood sugar control.


It is widely agreed that there is insuf-ficient information available on whichto base an RDA for pantothenic acid.Most countries that makerecommendations therefore give anestimate of safe and adequate levelsfor daily intake. These adequateintake levels (AI) are based on esti-mated dietary intakes in healthy pop-ulation groups and range, dependingon the health authority concerned,from 2 to 14 mg for adults.

Safety

Pantothenic acid is essentially consid-ered to be nontoxic, and no cases ofhypervitaminosis have ever beenreported. As much as 10 g daily inhumans produces only minor gastroin-testinal disturbance (diarrhoea). Due tothe lack of reports of adverse effectsthe main regulatory authorities havenot defined a tolerable upper level ofintake (UL) for pantothenic acid.

Supplements, foodfortification andcosmetics

Pure pantothenic acid is a viscoushygroscopic oil that is chemically notvery stable. Supplements thereforeusually contain the calcium salt, oralcohol, panthenol. Both are highlywater soluble and are rapidly convert-ed to free acid in the body. Calciumpantothenate is often included in mul-tivitamin preparations; panthenol isthe more common form used in mono-preparations, which are available in awide variety of pharmaceutical forms(e.g. solutions for injection and local

application, aerosols, tablets, oint-ments and creams). Pantethine, aderivative of pantothenic acid, is usedas a cholesterol and triglyceride-low-ering drug in Europe and Japan and isavailable in the U.S. as a dietary sup-plement.Pantothenate is added to a variety offoods, the most important of whichare breakfast cereals and beverages,dietetic and baby foods.D-Panthenol is often used in cosmeticproducts. In skin care products, ithelps to keep the skin moist and sup-ple, stimulates cell growth and tissuerepair, and inhibits inflammation andreddening. As a moisturiser and con-ditioner in hair care products, it pro-tects against and repairs damage dueto chemical and mechanical proce-dures (brushing, combing, shampoo-ing, perming, colouring etc.), andimparts sheen and luster.


Pantothenic acid is chemicallysynthesised by condensation of D-pantolactone with P-alanine.Addition of a calcium salt producescolourless crystals of calcium pan-tothenate. Panthenol is produced asa clear, almost colourless, viscoushygroscopic liquid.

78

History

1931 Williams and Truesdail separate an acid fraction from “bios”, thegrowth factor for yeast discovered in 1901 by Wildiers.

1933 Williams and coworkers show this fraction to be a single acidsubstance essential for the growth of yeast. Because they find itin a wide range of biological materials, they suggested calling it“pantothenic acid”.

1938 Williams and associates establish the structure of pantothenicacid

1939 Jukes and colleagues show the similarity between pantothenicacid and the chick antidermatitis factor.

1940 Total synthesis of the vitamin is achieved independently byWilliams and Major, Stiller and associates, Reichstein andGrüssner, and Kuhn and Wieland.

1947 Lipmann and his associates identify pantothenic acid as one ofthe components of the coenzyme they had discovered in livertwo years earlier.

1953 The full structure of coenzyme A is elucidated by Baddiley andcolleagues. Lipmann receives the Nobel Prize, together withKrebs, for his work on coenzyme A and its role in metabolism.

1954 Bean and Hodges report that pantothenic acid is essential inhuman nutrition. Subsequently, they and their colleaguesconduct several further studies to produce deficiency symptomsin healthy humans using the antagonist omega-methylpantothenic acid.

1965 Pugh and Wakil identify the acyl carrier protein as an additionalactive form of pantothenic acid.

1976 Fry and her associates measure the metabolic response ofhumans to deprivation of pantothenic acid without involvementof an antagonist. Fritz Albert Lipmann

Tadeusz Reichstein

Roger John Williams

79

Folic Acid

SynonymsFolacin, vitamin BC, vitamin B9, Lactobacillus casei factor

ChemistryFolic acid consists of a pteridine ring system, p-aminobenzoic acid and onemolecule of glutamic acid (chemical name: pteroylglutamic acid). Naturallyoccurring folates are pteroylpolyglutamic acids with two to eight glutamicacid groups.

NH2N

N

N

OH

N CH2

NH CO – NH – CH – CH2– COOH

COOH

Molecular formula of folic acid

Folic acid crystals in polarised light

80

IntroductionFolate is a generic term for a water-soluble group of B vitamins includingfolic acid and naturally occurringfolates. Folic acid is a syntheticfolate compound used in vitaminsupplements and fortif ied foodbecause of its increased stability.The name comes from folium, whichis the Latin word for leaves, becausefolates were first isolated fromspinach in 1941. In 1962 Herbertconsumed a folate-deficient diet forseveral months and records hisdevelopment of deficiency symp-toms. His findings set the criteria forthe diagnosis of folate deficiency.

Functions

Tetrahydrofolic acid, which is theactive form of folate in the body,acts as a coenzyme in numerousessential metabolic reactions. Folatecoenzymes act as acceptors anddonors of one-carbon units in thesereactions. Folate coenzymes play animportant role in the metabolism ofseveral amino acids, the con-stituents of proteins. The synthesisof the amino acid methionine fromhomocysteine requires a folatecoenzyme and, in addition, vitaminB12. Tetrahydrofolic acid is involvedin the synthesis of nucleic acids(DNA and RNA) – the molecules thatcarry genetic information in cells –and also in the formation of bloodcells. Folates are therefore essentialfor normal cell division, propergrowth and for optimal functioning ofthe bone marrow.

Main functions in a nutshell:• Coenzyme in amino acid metab-

olism • Coenzyme in the synthesis of

nucleic acids• Blood cell formation in the bone

marrow

Dietary sourcesFolates are found in a wide variety offoods. Its richest sources are liver,dark green leafy vegetables, beans,wheat germ and yeast. Othersources are egg yolk, milk and dairyproducts, beets, orange juice andwhole wheat bread.Folates synthesised by intestinalbacteria do not contribute signifi-cantly to folate nutrition in humansbecause bacterial folate synthesis isusually restricted to the large intes-tine (colon), whereas absorptionoccurs mainly in the upper part ofthe small intestine (jejunum).


Most dietary folates exist as polyglu-tamates, which have to be convert-ed to the monoglutamate form in thegut before absorption. The monoglu-tamate form is absorbed in the prox-imal small intestine by an active car-rier-mediated transport mechanism,and also by passive diffusion.Ingested folic acid is enzymaticallyreduced and methylated in themucosa cells. The predominant form

of folate in the plasma is 5-methylte-trahydrofolate.Folates are widely distributed in tis-sues, most of them as polyglutamatederivatives. The main storage organis the liver, which contains about halfof the body's stores.

BioavailabilityAbsorption of folic acid is almost100% when consumed underfasting conditions. When folic acidis consumed with a portion offood, bioavailability is estimatedfrom experimental data to be85%. The bioavailability of foodfolates is variable and incomplete,and has been estimated to be nomore than 50% that of folic acid.

Measurement

Different methods are used for themeasurement of folates. They canbe measured by microbiologicalassays using Lactobacillus casei astest organism. Radioassays basedon competitive protein binding aresimpler to perform and are notaffected by antibiotics, which givefalse low values in microbiologicalassays. High-performance liquidchromatography (HPLC) methodshave also been established for theanalysis of folates.Folate status is assessed by meas-uring serum and red blood cell folatelevels of methyltetrahydrofolate,which is the predominant folate.Serum folate level is not a reliableindicator of folate deficiency, but isconsidered a sensitive indicator ofrecent folate intake. Serum concen-trations < 7 nmol/L (3 ng/ml) aresuggested to indicate negative folatebalance. Levels in the red bloodcells are considered to be an indica-tor of long-term status, and to berepresentative of tissue folatestores. Levels < 305 nmol/L (140ng/ml) indicate inadequate folate

Food Folate (µg/100g)

Beef liver 592

Peanuts 169

Spinach 145

Broccoli 114

Asparagus 108

Egg 67

Strawberries 43

Orange juice

(freshly squeezed) 41

Tomatoes 22

Milk (whole) 6.7

Folate content of foods


81

status. A recent development hasbeen a method for the measurementof whole blood cell folate in driedblood spots on filter paper.Increased homocysteine levels mayalso indicate folate deficiency.Methyltetrahydrofolate is necessaryfor the conversion of homocysteineto methionine. Therefore plasmahomocysteine concentration in-creases when folate is not availablein sufficient amounts. Although plas-ma homocysteine concentration is asensitive indicator, it is not highlyspecific because it may be influ-enced by other nutrient deficiencies(vitamin B12, B6), genetic abnormal-ties and renal insufficiency.

Stability

Most forms of folate in food areunstable. Fresh leafy vegetablesstored at room temperature maylose up to 70% of their folate activi-ty within three days. Considerablelosses also occur through leachinginto cooking water (up to 95%) andthrough heating.

Interactions

Positive interactionsProper folate utilisation depends onan adequate supply of other vita-mins of the B group such as vitaminB12 and B6 and vitamin C, which areinvolved in the chemical reactionsneeded for folate metabolism.Vitamin C may also provide thereducing conditions needed to pre-serve folates in the diet, and a dietdeficient in folates is also likely to bedeficient in vitamin C.

Negative interactionsSeveral chemotherapeutic agents(e.g. methotrexate, trimethoprim,pyrimethamine) inhibit the enzymedihydrofolate reductase, which is nec-essary for the metabolism of folates.

When nonsteroidal anti-inflammato-ry drugs (e.g., aspirin, ibuprofen)are taken in very large thera-peutic doses, for example inthe treatment of severearthritis, they may interferewith folate metabolism.Many drugs may interfere withthe absorption, utilisationand storage offolates. These includealcohol, cholestyra-mine and colestipol (drugsused to lower blood choles-terol), antiepileptic agentssuch as barbiturates and diphenyl-hydantoin, and sulfasalazine, whichis used in the treatment of ulcerativecolitis. Drugs that reduce acidity inthe intestine, such as antacids andmodern anti-ulcer drugs, have alsobeen reported to interfere with theabsorption of folic acid.Early studies of oral contraceptivescontaining high levels of oestrogensuggested an adverse effect onfolate status, but this has not beensupported by more recent studieson low dose oral contraceptives.

Deficiency

Folate deficiency is one of the com-monest vitamin deficiencies. It canresult from inadequate intake, defec-tive absorption, abnormal metabo-lism or increased requirements. Diagnosis of a subclinical deficiencyrelies on demonstrating reduced redcell folate concentration or on otherbiochemical evidence such asincreased homocysteine concentra-tion, since haematological manifes-tations are usually absent. Earlysymptoms of folate deficiency arenon-specific and may include tired-ness, irritability and loss of appetite.Severe folate deficiency leads tomegaloblastic anaemia, a conditionin which the bone marrow producesgiant, immature red blood cells. Atan advanced stage of anaemiasymptoms of weakness, fatigue,

shortness of breath, irritabil ity,headache, and palpitations appear.If left untreated, megaloblasticanaemia may be fatal.Gastrointestinal symptoms alsoresult from severe folate deficiency.Deficiency during pregnancy mayresult in premature birth, infant lowbirth weight and foetal growth retar-dation. In children, growth may beretarded and puberty delayed.

Folate deficiency is very common inmany parts of the world and is partof the general problem of undernutri-tion. In developed countries, nutri-tional folate deficiency may beencountered above all in economi-cally underprivileged groups (e.g.,the elderly). Reduced folate intake isalso often seen in people on specialdiets (e.g. weight-reducing diets).Disorders of the stomach (e.g.atrophic gastritis) and small intestine(e.g. celiac disease, sprue, Crohn'sdisease) may lead to folate deficien-cy as a result of malabsorption. Inconditions with a high rate of cellturnover (e.g. cancer, certainanaemias and skin disorders), folaterequirements are increased. This isalso the case during pregnancy andlactation, due to rapid tissue growthduring pregnancy and to lossesthrough the milk during lactation.People undergoing drug treatment,e.g. for epilepsy, cancer or an infec-tion, are at high risk of developing a

82

folate deficiency, as are patients withrenal failure who require regularhaemodialysis. Acute folate defi-ciences have been reported to occurwithin a relatively short time inpatients undergoing intensive care,especially those on total parenteralnutrition.


In situations where there is a highrisk of folate deficiency, oral folicacid supplementation is recom-mended, usually in a multivitaminpreparation containing 400-500 µgof folic acid.In acute cases of megaloblasticanaemia, treatment often has to bestarted before a diagnosis of thecause (vitamin B12 or folate deficien-cy) has been made. To avoid compli-cations that may arise by treating aB12 deficiency with folic acid in suchcircumstances (see below), bothfolic acid and vitamin B12 need to beadministered until a specific diagno-sis is available.It has been demonstrated that peri-conceptional (before and during thefirst 28 days after conception) sup-plementation of women with folicacid can decrease the risk of neuraltube defects (malformations of thebrain and spinal cord, causing anen-cephaly or spina bifida). Therefore, adaily intake of 400 µg folic acid inaddition to a healthy diet 8 weeksprior to and during the first 12 weeksafter conception is recommended.There is evidence that adequatefolate status may also prevent theincidence of other birth defects,including cleft lip and palate, certainheart defects and limb malforma-tions.Results from intervention studieshave shown that a multivitamin sup-plement containing folic acid is more

effective in decreasing the risk ofneural tube defects and other birthdefects than folic acid alone.Numerous studies have shown thateven moderately elevated levels of homocysteine in the blood increasethe risk of atherosclerosis. Folic acidhas been shown to decrease homo-cysteine levels. Several randomisedplacebo-controlled trials arepresently being conducted to estab-lish whether folic acid supplementa-tion reduces the risk of cardiovascu-lar diseases by lowering homocys-teine blood levels.A number of different observationalstudies have found poor folate sta-tus to be associated with increasedcancer risk. There is evidence thatfolate plays a role in preventing col-orectal cancer. The results of twolarge epidemiological investigationssuggest that increased folate intakemay reduce breast cancer risk asso-ciated with regular alcohol con-sumption.Low folate levels have also beenassociated with Alzheimer´s disease,dementia and depression.


In the USA the recommendations ofthe Food and Nutrition Board (1998)are expressed as DFEs. This organi-sation recommends a daily intake of400 µg of DFE for adult females andmales. To cover increased needsduring pregnancy and lactation, itrecommends 600 µg/day and 500µg/day respectively. In Europe, theRDA varies between 200-400µg/day for adults in different coun-tries.

Dietary Folate Equivalents (DFE)have been introduced because ofthe different bioavailabil ity offolates and folic acid.

1 µg DFE = 1 µg of food folate= 0.5 µg of folic acid

taken on an emptystomach

= 0.6 µg of folic acidfrom fortified food oras a supplementtaken with meals


RDA*

RDA listed as dietary folate

Infants , 6 months 65 µg (AI)

Infants 7-12 months 80 µg (AI)

Children 1-3 years 150 µg



Adults . 14 years 400 µg

Pregnancy 600 µg

Lactation 500 µg












83

Safety

Oral folic acid is not toxic tohumans. Even with daily doses ashigh as 15 mg there have been nosubstantiated reports of toxicity, anda daily supplement of 10 mg hasbeen taken for five years withoutadverse effect. It has been claimed that high dosesof folic acid may counteract theeffect of antiepileptic medicationand so increase the frequency ofseizures in susceptible patients.A high intake of folic acid can maskvitamin B12 deficiency. It shouldtherefore not be used indiscriminate-ly in patients with anaemia becauseof the risk of damage to the nervoussystem due to B12 deficiency.The US Food and Nutrition Board(1998) set the tolerable upper intakelevel (UL) of folic acid from fortifiedfoods or supplements at 1,000µg/day for adults. The EU ScientificCommittee on Food (2000) alsoestablished a UL of 1,000 µg for folicacid.


Folic acid is available as oral prepa-rations, alone or in combination withother vitamins or minerals (e.g. iron),and as an aqueous solution forinjection. As the acid is only poorlysoluble in water, folate salts areused to prepare liquid dosageforms. Folinic acid (also known asleucovorin or citrovorum factor) is aderivative of folic acid administeredby intramuscular injection to circum-vent the action of dihydrofolatereductase inhibitors, such asmethotrexate. It is not otherwiseindicated for the prevention or treat-ment of folic acid deficiency.

Folic acid is added to a variety offoods, the most important of whichare flour, salt, breakfast cereals andbeverages, soft drinks and babyfoods.

To reduce the risk of neural tubedefects, cereal grains are fortifiedwith folate in some countries. Inthe USA and Canada all enrichedcereal grains (e.g., enrichedbread, pasta, flour, breakfastcereals, and rice) are required tobe fortif ied with folic acid. InHungary and Chile, wheat flour isfortified with folic acid.


Folic acid is manufactured on alarge scale by chemical synthesis.Various processes are known. Mostsynthesised folic acid is used in ani-mal feed.

Robert B. Angier

Tom Spies

Esmond Emerson Snell

84

History

1931 Wills in India observes the effect of liver and yeast extracts on tropicalmacrocytic anaemia and concludes that this disorder must be due to adietary deficiency. She recognises that yeast contains a curative agentequal in potency to that of liver.

1938 Day and coworkers find an antianaemia factor for monkeys in yeast anddesignate it “vitamin M.” Around the same time, Stokstad and Manningdiscover a growth factor for chicks, which they call “Factor U”.

1939 Hogan and Parrott identify an antianaemia factor for chicks in liverextracts, which they name “Vitamin BC”.

1940 Discovery of growth factors for Lactobacillus casei and Streptococcuslactis. Snell and Peterson coin the term “norite-eluate factor”.

1941 Mitchell and colleagues suggest the name “folic acid” (folium, Latin forleaf) for the factor responsible for growth stimulation of Streptococcuslactis, which they isolate from spinach and suspect of having vitamin-likeproperties for animals.

1945 Angier and coworkers report the synthesis of a compound identical to theL. casei factor isolated from liver. They later describe the chemical struc-tures of the basic and related compounds.

1945 Spies demonstrates that folic acid cures megaloblastic anaemia duringpregnancy.

1962 Herbert consumes a folate-deficient diet for several months and recordshis development of deficiency symptoms. His findings set the criteria forthe diagnosis of folate deficiency. In the same year, Herbert estimates thefolic acid requirements for adults, which still serve as a basis for manyRDAs.

1991 Wald establishes that folic acid supplementation reduces risk of neuraltube defects by 70% among women who have already given birth to achild with such birth defects.

1992 Butterworth finds that higher than normal serum levels of folic acid areassociated with decreased risk of cervical cancer in women infected withhuman papillomavirus. Also, Czeizel demonstrates that first-time occur-rence of neural tube defects may be largely eliminated with a multivitamincontaining folic acid taken in the periconceptional period.

1993 The US Public Health Service recommends that all women of childbearingpotential consume 0.4 mg (400 µg) of folate daily in order to reduce the riskof foetal malformations such as spina bifida and other neural tube defects.

1998 Fortification of all enriched cereal grains (e.g., enriched bread, pasta, flour,rice and breakfast cereals) with folic acid becomes mandatory in the USAand in Canada. In Hungary, wheat flour is fortified with folic acid.

2000 Fortification of wheat flour with folic acid is established in Chile.

85

Biotin

H

ON

COOH

NHH

S

HH

Molecular formula of biotin

SynonymsVitamin H (“Haar und Haut”, German words for “hair and skin”), vitamin B8and co-enzyme R.

ChemistryBiotin has a bicyclic ring structure. One ring contains a ureido group and theother contains a heterocyclic sulphur atom and a valeric acid side-group.(Hexahydro-2-oxo-1H-thieno [3,4-d]imidazole-4-pentanoic acid). Bio-logically active analogues: biocytin (e-N-biotinyl-L-lysine), oxybiotin (S sub-stituted with O).Biotin crystals in polarised light

86

IntroductionBiotin is a colorless, water-solublemember of the B-complex group ofvitamins. Although biotin was dis-covered already in 1901 as a specialgrowth factor for yeast, it took near-ly forty years of research to establishbiotin as a vitamin. Due to its bene-ficial effects for hair, skin and nails,biotin is also known as the “beautyvitamin”. There are eight differentforms of biotin, but only one of them– D-biotin – occurs naturally and hasfull vitamin activity. Biotin can onlybe synthesised by bacteria, moulds,yeasts, algae, and by certain plantspecies.

Functions

Biotin plays a key role in the metab-olism of lipids, proteins and carbo-hydrates. It acts as a critical coen-zyme of four carboxylases(enzymes):

• acetyl-CoA carboxylase (involvedin the synthesis of fatty acids fromacetate)

• propionyl-CoA carboxylase (in-volved in gluconeogenesis, i.e. thegeneration of glucose from lactate,glycerol, and amino acids)

• b-methylcrotonyl-CoA carboxylase(necessary for the metabolism ofleucin, an essential amino acid)

• pyruvate carboxylase (involved inenergy metabolism, necessary forthe metabolism of amino acids,cholesterol, and odd chain fattyacids)

Biotin also plays a special role inenabling the body to use blood glu-cose as a major source of energy forbody fluids. Furthermore, biotin mayhave a role in DNA replication andtranscription arising from its interac-tion with nuclear histone proteins. Itowes its reputation as the “beautyvitamin” to the fact that it activatesprotein/amino acid metabolism inthe hair roots and fingernail cells.

Main functions in a nutshell:• Synthesis of fatty acids, amino

acids and glucose• Energy metabolism• Excretion of by-products from

protein metabolism• Maintenance of healthy hair,

toenails and fingernails

Dietary sources

Biotin is widely distributed in mostfoods but at very low levels com-pared to other water-soluble vita-mins. It is found in free and protein-bound forms in foods. Its richestsources are yeast, liver and kidney.Egg yolk, soybeans, nuts and cere-als are also good sources. 100 g ofliver contains approximately 100 µgbiotin, whereas most other meats,vegetables and fruits only containapproximately 1 µg biotin /100 g. Inanimal experiments, biotin bioavail-ability has been shown to vary con-siderably (5%-62%), and in cereals itappears to be lower.

Biotin-producing microorganismsexist in the large intestine, but theextent and significance of this enter-

al synthesis in the overall biotinturnover is difficult to calculate andthus remains a subject of controver-sy.


In most foodstuffs biotin is bound toproteins from which it is released inthe intestine by protein hydrolysisand a specific enzyme, biotinidase.Biotin is then absorbed unchangedin the upper part of the small intes-tine by an electron-neutral sodium(Na+) gradient dependent carrier-mediated process and also by slowpassive diffusion. The carrier is reg-ulated by the availability of biotin,with up-regulation of the number oftransporter molecules when biotin isdeficient. The colon is also able toabsorb biotin via an analogue trans-port mechanism. Once absorbed,biotin is distributed to all tissues.The presence of a specific biotincarrier protein in plasma is not yetconclusive. The liver and retinal tis-sues are the main storage places.Biotin metabolites are not active asvitamins and are excreted in theurine. Remarkable amounts of biotinappear in the faeces deriving fromcolonic bacteria.

Measurement

The body status of biotin can bedetermined by measuring its activityand/or activation of biotin depen-dent enzymes – predominately car-boxylases – by added biotin. Moreconvenient methods are directdetermination of biotin in plasma orserum by microbiological methodsor avidin binding assays, or determi-nation of biotin excretion and 3-hydroisovaleric acid in urine.Measurement of biotin in plasma isnot a reliable indicator of nutritional

Food Biotin(µg/100g)

Brewer’s yeast 115

Beef liver 100

Soya beans 60

Wheat bran 45

Peanuts 35

Egg 25

White mushrooms 16

Spinach 6.9

Bananas 6

Strawberries 4

Whole wheat bread 2

Asparagus 2

Biotin content of foods


87

status, because reported levels forbiotin in the blood vary widely. Thus,a low plasma biotin concentration isnot a sensitive indicator of inade-quate intake.Usual serum concentrations = 100 -400 pmol/L.

Stability

Biotin is relatively stable when heat-ed and so is not easily destroyed inthe ordinary processes of cookingbut it will leach into cooking water.Processing of food, e.g. canning,causes a moderate reduction inbiotin content.

Interactions

Negative interactionsRaw egg whites contain avidin, aglycoprotein that strongly binds withbiotin and prevents its absorption.Thus, the ingestion of large quanti-ties of raw egg white over a longperiod can result in a biotin deficien-cy. It has also been reported thatantibiotics which damage the intes-tinal flora (thus decreasing bacterialsynthesis) can reduce biotin levels.Interactions with certain anticonvul-sant drugs and alcohol have alsobeen reported, as they may inhibitintestinal carrier-mediated transportof biotin. Pantothenic acid ingestedin large amounts competes withbiotin for intestinal and cellularuptake because of their similarstructures.

Deficiency

Human biotin deficiency is extremelyrare. This is probably due to the factthat biotin is synthesised by benefi-cial bacteria in the human intestinaltract. Potential deficiency symptomsinclude anorexia, nausea, vomiting,glossitis, depression, dry scaly der-matitis, conjunctivitis and ataxia,and after long-lasting, severe biotindeficiency, loss of hair colour andhair loss (alopecia). Signs of biotindeficiency in humans have beendemonstrated in volunteers consum-ing a biotin-deficient diet togetherwith large amounts of raw egg white.After 3-4 weeks they developed afine dry scaly desquamating der-matitis, frequently around the eyes,nose, and mouth. After ten weeks onthe diet, they were fatigued,depressed and sleepy, with nauseaand loss of appetite. Muscular pains,hyperesthesia and paresthesiaoccurred, without reflex changes orother objective signs of neuropathy.Volunteers also developed anaemiaand hypercholesterolaemia. Liverbiopsies in sudden infant deathsyndrome babies reveal low biotinlevels. Most of the affected infantswere bottle-fed.

Groups at risk of deficiency• patients maintained on total

parenteral nutrition• people who eat large amounts of

raw egg white• haemodialysis patients• diabetes mellitus• individuals receiving some forms of

long-term anticonvulsant therapy• individuals with biotinidase defi-

ciency or holocarboxylase syn-thetase (HCS) deficiency (geneticdefects)

• patients with malabsorption,including short-gut syndrome

• pregnancy may be associated withmarginal biotin deficiency

Disease prevention andtherapeutic useThere is no direct evidence that mar-ginal biotin deficiency causes birthdefects in humans, but an adequatebiotin intake/supplementation duringpregnancy is advisable.Biotin is used clinically to treat thebiotin-responsive inborn errors ofmetabolism, holocarboxylase syn-thetase deficiency and biotinidasedeficiency.Large doses of biotin may be givento babies with a condition calledinfantile seborrhea or to patientswith genetic abnormalities in biotinmetabolism. A large number ofreports have shown a beneficialeffect of biotin in infant seborrheicdermatitis and Leiner's disease (ageneralised form of seborrheic der-matitis).Biotin supplements are sometimesgiven to help reduce blood sugar indiabetes patients. People with type2 diabetes often have low levels ofbiotin. Some patients with diabetesmay have an abnormality in thebiotin-dependent enzyme pyruvatecarboxylase, which can lead to dys-function of the nervous system. The main benefit of biotin as a

88

dietary supplement is in strengthen-ing hair and nails. Biotin supple-ments may improve thin or splittingtoenails or fingernails and improvehair health. Uncomable hair syn-drome in children also improves withbiotin supplementation, as do cer-tain skin disorders, such as “cradlecap”. Biotin has also been used tocombat premature graying of hair,though it is likely to be useful onlyfor those with a low biotin status. Inorthomolecular medicine biotin isused to treat hair loss, but scientificevidence is not conclusive.Biotin has been used for people inweight loss programs to help themmetabolise fat more efficiently.


In 1998 the Food and NutritionBoard of the Institute of Medicinefelt the existing scientific evidencewas insufficient to calculate an EAR,

and thus an RDA, for biotin. Insteadan Adequate Intake level (AI) hasbeen defined. The AI for biotinassumes that current averageintakes of biotin (35 µg to 60 µg/day)are meeting the dietary requirement.An estimation of the safe and ade-quate daily dietary intake for biotinwas made for the first time in 1980by the Food and Nutrition Board ofthe United States National ResearchCouncil. The present recommenda-tions in the USA are 20-30 µg dailyfor adults and children over 11years, and 5-12 µg daily for infantsand younger children. France andSouth Africa recommend a dailyintake of up to 300 µg, andSingapore up to 400 µg biotin.Others, including the FederalRepublic of Germany, assume thatdiet and intestinal synthesis providesufficient amounts.

Safety

No known toxicity has been associ-ated with biotin. Biotin has beenadministered in doses as high as 40

mg per day without objectionableeffects. Due to the lack of reports ofadverse effects, no major regulatoryauthorities have established a toler-able upper level of intake (UL) forbiotin.


Biotin, usually either in the form ofcrystall ine D-biotin or brewer’syeast, is added to many dietary sup-plements, infant milk formulas andbaby foods, as well as variousdietetic products. As a supplement,biotin is often included in combina-tions of the B vitamins. Mono-preparations of biotin are available insome countries as oral and par-enteral formulations.Therapeutic doses of biotin forpatients with a biotin deficiencyrange between 5 and 20 mg daily.Seborrheic dermatitis and Leiner'sdisease in infants respond to dailydoses of 5 mg. Patients with bio-tinidase deficiency require life-long


RDA*

Infants , 6 months 5 µg (Adequate Intake, AI)

Infants 7-12 months 6 µg (AI)





Adults . 19 years 30 µg (AI)

Pregnancy 30 µg (AI)

Lactation 35 µg (AI)












89

biotin therapy in milligram doses (5-10mg/day). Patients with HCS defi-ciency require supplementation of40-100 mg/day. If biotin therapy isintroduced in infancy, the prognosisfor both these genetic defects aregood.A daily supplement of 60 µg biotinfor adults and 20 µg for children hasbeen recommended to maintain nor-mal plasma levels in patients on totalparenteral nutrition.

Other technicalapplications

Baker's yeast (Saccharomycescerevisiae) is dependent on biotinfor growth. Biotin is therefore addedas a growth stimulant to the nutrientmedium used in yeast fermentation.Also, many of the microorganismsused in modern biotechnology arebiotin-dependent. Thus, biotin isadded to the growth medium insuch cases.

In cosmetics, biotin is used as aningredient for hair care products.


Commercial synthesis of biotin isbased on a method developed byGoldberg and Sternbach in 1949and using fumaric acid as startingmaterial. This technique produces apure D-biotin which is identical tothe natural product.

Vincent du Vigneaud

Feodor Lynen

Fritz Kögl

Paul György

90

History

1901 Wildiers discovers that yeast requires a special growth factorwhich he names “bios”. Over the next 30 years, bios proves to bea mixture of essential factors, one of which – bios IIB – is biotin.

1916 Bateman observes the detrimental effect of feeding high doses ofraw egg white to animals.

1927 Boas confirms the findings of dermatosis and hair loss in rats fedwith raw egg white. She shows that this egg white injury can becured by a “protective factor X” found in the liver.

1931 György also discovers this factor in the liver and calls it vitamin H(from Haut, the German word for skin).

1933 Allison and coworkers isolate a respiratory coenzyme – coenzymeR – that is essential for the growth of Rhizobium, a nitrogen-fixingbacterium found in leguminous plants.

1935 Kögl and Tönnis extract a crystalline growth factor from dried eggyolk and suggest the name ‘biotin’.

1940 György and his associates conclude that biotin, vitamin H andcoenzyme R are identical. They also succeed in isolating biotinfrom the liver.

1942 Kögl and his group in Europe and du Vigneaud and his associatesin the USA establish the structure of biotin.

1942 Sydenstricker and colleagues demonstrate the need for biotin inthe human diet.

1943 Total synthesis of biotin by Harris and colleagues in the USA.

1949 Goldberg and Sternbach develop a technique for the industrialproduction of biotin.

1956 Traub confirms the structure of biotin by X-ray analysis.

1959 Lynen's group describes the biological function of biotin andpaves the way for further studies on the carboxylase enzymes.

1971 First description of an inborn error of biotin-dependent carboxy-lase metabolism by Gompertz and associates.

1981 Burri and her colleagues show that the early infantile form ofmultiple carboxylase deficiency is due to a mutation affectingholocarboxylase synthetase activity.

1983 Wolf and coworkers suggest that late-onset multiple carboxylasedeficiency results from a deficiency in biotinidase activity.

91

Dietary Reference Intakes for Cal-cium, Phosphorus, Magnesium,Vitamin D, and Fluoride (1997)National Academy of Sciences.Institute of Medicine. Food andNutrition Board.

Dietary Reference Intakes forThiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12,Pantothenic Acid, Biotin, andCholine (1998) National Academy of Sciences. Institute of Medicine.Food and Nutrition Board.

Dietary Reference Intakes forVitamin C, Vitamin E, Selenium, and Carotenoids (2000) NationalAcademy of Sciences. Institute ofMedicine. Food and Nutrition Board.

Dietary Reference Intakes forVitamin A, Vitamin K, Arsenic,Boron, Chromium, Copper, Iodine,Iron, Manganese, Molybdenum,Nickel, Silicon, Vanadium, and Zinc(2001) National Academy ofSciences. Institute of Medicine.Food and Nutrition Board.

ht tp: / / r i ley.na l .usda.gov/na l_d is-play/index.php?info_center=4&tax_level=3&tax_subject=256&topic_id=1342&level3_id=5141

The Linus Pauling Institute, Micro-nutrient Information Center.http://lpi.oregonstate.edu/

Hidgon, J. An evidence-basedapproach to vitamins and minerals.Health benefits and intake recom-mendations. Thieme, 2003.

Bowman, BA, Russell, RB (eds.)Present Knowledge in Nutrition, 8th edition ILSI Press Washingtion,DC; 2001.

Expert Group on Vitamins andMinerals. Safe upper levels for vita-mins and minerals, Food StandardsAgency, United Kingdom, 2003.http://www.foodstandards.gov.uk/multimedia/pdfs/vitmin2003.pdf

D-A-CH (2000) Deutsche Gesell-schaft für Ernährung (DGE), Österreichische Gesellschaft für Ernährung (ÖGE), SchweizerischeGesellschaft für Ernährung (SGE),Schweizerische Vereinigung fürErnährung (SVE): Referenzwerte für die Nährstoffzufuhr. 1. Aufl.,UmschauBraus, Frankfurt/Main.

Souci Fachmann Kraut, FoodComposition and Nutrition Tables,6th ed, CRC Press, Boca Raton,2000.http://www.sfk-online.net

Vitamin and mineral requirements inhuman nutrition. 2nd ed. WorldHealth Organization and Food andAgriculture Organization of theUnited Nations, 2004.

Brody T. Nutritional Biochemistry.2nd ed. San Diego: Academic Press;1999.

Opinions of the Scientific Committeeon Food on the Tolerable UpperIntake Levels of vitamins andmineralshttp://europa.eu.int/comm/food/fs/sc/scf/index_en.html

References

92

measurement 19negative interactions 19provitamin A 18RDA 20RE (retinol equivalent) 19safety 20skin 19stability 19supplements and food fortification 20

carotenodermia 20cholestyramine 19colestipol 19dioxygenase 18free radicals 18HPLC (high performance liquidchromatography) 19lipids 18omeprazole 19orlistat 19prostaglandin synthesis 18provitamin A carotenoids 18, 19singlet oxygen 18terpene 17

Vitamin D

alkaline phosphatase 25anticonvulsant 25antirachitic factor 23, 27bone density 25calcidiol (25-hydroxycholecalciferol) 24calcitriol (1,25-dihydroxy-cholecalciferol) 24, 27calcium 24, 25, 26cell proliferation 24cholecalciferol (vitamin D3) 24cholestyramine 25corticosteroid hormones 25diabetes mellitus 25differentiation 24, 27ergocalciferol (vitamin D2) 24hypervitaminosis D 26hypovitaminosis D 25insulin secretion 24laxatives 25mineral balance 24

Vitamin A

acne 14axerophthol 11beta-carotene 18bone mineral density 15carotenoids 11, 13, 18chylomicrons 13cornea 12, 14differentiation 12embryonic development 12enterocytes 13epithelial cells 12erythropoiesis 13follicular hyperkeratosis 14gene expression 12hormone 12hypervitaminosis A 15immune system 12iron 13killer cells 13lymphocytes 13malformations 14, 15measles 13, 14nuclear receptor proteins 12opsin 12phagocytes 13provitamin A 13psoriasis 14reproduction 12retinal 12, 15retinoic acid 12, 16retinol 12, 13, 15, 16retinol equivalent (RE) 13retinyl esters 11, 12rhodopsin 12rod cells 11-16vitamin A 3–8see also beta-carotene

blindness 12bone health 15cellular differentiation 12chemistry 11deficiency 12dietary sources 13disease prevention and therapeutic use 12functions 12growth and development 12

history 16immune function 12industrial production 15introduction 12measurement 13mortality 14negative interactions 13positive interactions 13pregnancy 14prevention of vitamin A deficiency 14RDA 14RE (retinol equivalent) 13safety 15stability 13supplements and food fortification 15UL 15vision 12vitamin A activity 13vitamin A content of foods 11vitamin E 13

vitamin A analogues 14xerophthalmia 14, 16zinc 13

beta-carotene

beta-carotene 17-22see also vitamin A

absorption and body stores 18antioxidant activity 18beta-carotene content of foods 18bioavailability 18cancer and cardiovascular diseases 19chemistry 17deficiency 19dietary sources 18disease prevention and therapeutic use 19erythropoietic protoporphyria 20free radical chain reactions 18functions 18history 21immune system 19industrial production 20introduction 18

Index

93

multiple sclerosis 25osteomalacia 25, 27osteoporosis 25parathyroid hormone (PTH 24, 25phosphorus 24, 25psoriasis 25, 27rheumatoid arthritis 25rickets 25, 27seco-steroid 23vitamin D 23-28

absorption and body stores 24autoimmune diseases 25bone formation and mineralisation 24calcium and phosphate blood levels 24cancer 25chemistry 23control of cell proliferation and differentiation 24deficiency 25dietary sources 24disease prevention and therapeutic use 25endogenous synthesis 24exposure to sunlight 25functions 24groups at risk of deficiency 25hereditary vitamin D-dependent rickets 25history 27immune system 24industrial production 26introduction 24measurement 24negative interactions 25positive interactions 25prevention of osteoporosis 25RDA 26safety 26stability 24supplements and food fortification 24UL 26vitamin D content of foods 24

vitamin D analogues 25vitamin D receptor (VDR) 24, 28vitamin D-dependent rickets 25

Vitamin E

Alzheimer´s disease 32amyotrophic lateral sclerosis 32anticoagulants 33

blood coagulation 31cell adhesion molecules 30cell proliferation 30, 32cholestyramine 31chylomicrons 31colestipol 31differentiation 30endothelial cells 30endothelium 30free radicals 30haemolytic anaemia 32HPLC (high performance liquidchromatography) 31intraventricular haemorrhage 32iron 31isoniazid 31lipid peroxidation 32lipoproteins 31low density lipoprotein (LDL) 30, 36myopathy 32neuropathy 32nitrosamines 33pigmented retinopathy 32platelet aggregation 30polyunsaturated fatty acids (PUFAs) 30, 31protein kinase C 30retrolental fibroplasia 32selenium 31tocopherol esters 31tocopherols 29, 30tocopheryl acetate 31tocopheryl succinate 31tocotrienols 29, 30vasodilation 30vitamin E 29-34

absorption and body stores 31atherosclerosis 32beta-carotene 31cancer 32cardiovascular diseases 32cataracts 32chemistry 29common cold 32deficiency 32dietary sources 30disease prevention and therapeutic use 32fat soluble antioxidant 30functions 30history 34immunity in the elderly 32industrial production 33introduction 30measurement 31

negative interactions 31neurodegenerative diseases 32non-antioxidant functions 30oxidative stress 32positive interactions 31RDA 32RRR-configuration 30safety 32stability 31supplements, food fortificationsand other applications 33UL 33vitamin C 30vitamin E content of foods 30vitamin K 31a-tocopherol equivalents (a-TE) 30a-tocopherol transfer protein 31

Vitamin K

antibiotics 37bacterial flora 36blood clotting 37calcification 36carboxylation 36, 37, 39cholestyramine 37clotting time 37colestipol 37coumarin anticoagulants 37fat malabsorption 37haemorrhages 36HPLC (high performance liquidchromatography) 37hypoprothrombinemia 37ileum 36intracranial haemorrhage 35, 36jaundice 38jejunum 36kernicterus 38matrix Gla-protein (MGP) 36menadione (vitamin K3) 38menaquinones (vitamin K2) 36, 37orlistat 37osteocalcin 36, 37phylloquinone (vitamin K1) 35, 36prothrombin (factor II) 36salicylates 37vitamin K 35-39

absorption and body stores 37animal nutrition 36antitumor activity 38atherosclerosis 38blood coagulation 36bone metabolism 36

94

chemistry 35coenzyme 36deficiency 37dietary sources 36disease prevention and therapeutic use 37functions 36history 39industrial production 38infant morbidity and mortality 37intestinal bacteria 37introduction 36measurement 37negative interactions 37newborn infants 37osteoporosis 38RDA 38safety 38stability 37supplements, food fortifications and other applications 38vitamin K antagonists 37vitamin K content of foods 36vitamin K cycle 37vitamin K-dependent proteins 36

Vitamin C

advanced age-related maculardegeneration (AMD) 41anti-scorbutic vitamin 40ascorbic acid 40bioflavonoids 42carnitine 41cholesterol 41, 43collagen 41galactose 41gangrene 42glucose 41, 42haemorrhages 42histamine 41neurotransmitters 41nitrosamines 41orthomolecular medicine 43peptide hormones 41petechiae 42plaque 43scurvy 41, 42, 45stress 41vitamin C 40-46

absorption and body stores 41antioxidant 41blood pressure 43cancer 43

cardiovascular diseases 43chemistry 40common cold 41deficiency 42dietary sources 41disease prevention and therapeutic use 43functions 41history 45industrial production 44introduction 41measurement 41negative interactions 42positive interactions 42RDA 43safety 44stability 42supplements and food fortification 44UL 44uses in food technology 44vitamin C content of foods 41vitamin C loss in foods 42vitamin E 41wound healing 43zinc 41

Vitamin B1

acetylcholin 48adrenalin 48aneurine see vitamin B1antineuritic factor see vitamin B1beriberi 48carbohydrate metabolism 48, 49HPLC (high performance liquidchromatography) 50neurotransmitters 48pentose phosphate pathway 48serotonin 48thiamin see vitamin B1thiamin diphosphate (TDP) 47thiamin monophosphate (TMP) 47thiamin pyrophoshate (TPP) 48thiamin triphosphate (TTP) 47thiaminases 49transketolase 48, 49vitamin B1 47-52

absorption and body stores 49antioxidant vitamins 49arsenic 48chemistry 48chronic alcoholism 50coenzyme 48

deficiency 49dietary sources 48disease prevention and therapeutic use 50functions 48history 52industrial production 51introduction 48measurement 49negative interactions 49niacin 49pantothenic acid 49positive interactions 49RDA 51safety 51stability 49supplements and food fortification 51vitamin B1 content of foods 48vitamin B12 49vitamin B6 49

Wernicke-Korsakoff syndrome 49, 50

Vitamin B2

anaemia 56antibiotics 55barbiturates 55calcium 56cataracts 56chlorpromazin 55creatinine 55depression 56FAD dependent erythrocyte glutathion reductase 55flavin adenine dinucleotide (FAD) 53flavin mononucleotide (FMN) 54, 55, 58flavoproteins 54, 55homocysteine 56HPLC (high performance liquidchromatography) 55iron 56lactoflavin 53migraine 57ouabain 55ovoflavin 53oxidation-reduction (redox) reactions 54peripheral neuropathy 56probenecid 55respiratory chain 54riboflavin see vitamin B2

95

theophylline 55thyroxine 55triiodothyroxine 55vitamin B2 53-58

absorption and body stores 54antioxidants 56chemistry 53coenzymes 54deficiency 55dietary sources 54disease prevention and therapeutic use 56eye-related diseases 56folic acid 54functions 54groups at risk of deficiency 56history 58industrial production 57introduction 54measurement 55migraines 57negative interactions 55positive interactions 55pyridoxine 54RDA 57safety 57stability 55supplements and food fortification 57vitamin B2 content of foods 54vitamin B-complex 54

zinc 56

Vitamin B6

asthma 63autism 63carpal tunnel syndrome 63Chinese restaurant syndrome 63depression 63diabetes mellitus 63erythrocyte alanine aminotransferase 61erythrocyte aspartate aminotransferase 61haemoglobin 60, 61, 62histamine metabolism 60homocysteine 60, 62, 63HPLC (high performance liquidchromatography) 61hydrochloric acid 60hyperemesis gravidarum 63hyperhomocystinaemia 63immune system 60, 62, 63

kidney stones 63, 64neurotransmitters 60premenstrual syndrome (PMS) 62prostaglandines 60pyridoxal see vitamin B6pyridoxal 5`-phosphate (PLP) 60pyridoxamine see vitamin B6pyridoxic acid 61pyridoxine see vitamin B6serotonin 60sideroblastic anaemias 62sodium-potassium balance 62tryptophan 60tryptophan load test 61vitamin B6 59-64

absorption and body stores 60chemistry 59coenzyme 60deficiency 61dietary sources 60disease prevention and therapeutic use 61functions 60history 63introduction 60magnesium 61measurement 61negative interactions 61positive interactions 61RDA 62riboflavin 60safety 62stability 61supplements and food fortification 62vitamin B12 60vitamin B6 content of foods 60vitamin B-complex 60zinc 60

xanthurenic acid 61

Vitamin B12

adenosylcobalamin 65antibiotics 67anticonvulsants 68cholestyramine 67cobalamin see vitamin B12cobalt 65colchicine 67corrinoids 66gastric atrophy 68homocysteine 66hydroxocobalamin 65, 70

hyperhomocysteinaemia 68intermediary metabolism 66intrinsic factor 67, 68, 70methionine synthase 66methylcobalamin 65methylmalonic acid 67methylmalonyl CoA mutase 66nerve sheaths 66nitrous oxide 68para-aminosalicylic acid 67passive diffusion 67pernicious anaemia 66, 68, 69, 70phagocytosis 67red blood cell formation 66Schilling test 67vegetarians 68vitamin B12 65-70

absorption and body stores 67cancer 69chemistry 65coenzyme 66deficiency 68dietary sources 66disease prevention and therapeutic use 68excretion 67folic acid 66functions 66history 70industrial production 69introduction 66measurement 67microbial synthesis 66negative interactions 67RDA 69safety 69stability 67supplements and food fortification 69UL 69vitamin B12 content of foods 66vitamin B6 67

Niacin

adenosine diphosphate (ADP)-ribose 72carcinoid syndrome 73cell differentiation 72cellular signal transduction 72copper 73diazepam 73DNA repair 72, 73DNA replication 72

96

facilitated diffusion 72glucose intolerance 74Hartnup’s disease 73hepatotoxicity 74high-density lipoprotein cholesterol 73HIV (human immunodeficiency virus) 73hyperglycaemia 74insulin-secreting b-cells 73iron 73isoniazid 73low-densitiy lipoprotein cholesterol 73niacin 71-75

absorption and body stores 72AIDS 73bioavailability 72cancer 73cardiovascular disease 73chemistry 71coenzymes 72deficiency 73diabetes mellitus 73dietary sources 72disease prevention and therapeutic use 73functions 72history 75industrial production 74introduction 72measurement 72NAD 72NADP 72negative interactions 73niacin content of foods 72niacin equivalent (NE) 74RDA 74riboflavin 73safety 74stability 73supplements and food fortification 74tryptophan 72UL 74vitamin B6 73

nicotinamide see niacinnicotinamide adenine dinucleotide(NAD) 72nicotinamide adenine dinucleotidephosphate (NADP) 72nicotinic acid see niacinN-methyl-2-pyridone-5-carboxamide 72N-methyl-nicotinamide 72

oxidation-reduction (redox) reactions 72pancreas 73passive diffusion 72pellagra 71, 72, 75penicillamine 73phenobarbitol 73phenytoin 73PP factor (pellagra-preventative factor) 71serotonin 73triacylglycerols 73tryptophan 72, 73, 74, 75tuberculosis 73

Vitamin B5

acyl carrier protein 77, 80alcohol detoxification 77calcium pantothenate 77, 79cholesterol 77coenyzme Q10 77homopantothenate 78hormones 77HPLC (high performance liquidchromatography) 77L-carnitine 77methyl bromide 78neurotransmitters 77omega-methyl pantothenic acid 78, 80pantetheine 76, 78pantethine 79panthenol 77, 79pantothenic acid 76-80

absorption and body stores 77biotin 78chemistry 76coenzyme A 77deficiency 78dietary sources 77disease prevention and therapeutic use 79enteral synthesis 77folic acid 78functions 77groups at risk of deficiency 78history 80industrial production 79introduction 77measurement 77negative interactions 78pantothenic acid content of foods 76

positive interactions 78RDA 79safety 79stability 78supplements, food fortification and cosmetics 79UL 79vitamin A 78vitamin B12 78vitamin B6 78vitamin C 78

passive diffusion 77phospholipids 77porphyrin 77sphingolipids 77sterols 77vitamin B5 see pantothenic acid

Folic Acid

amino acids 82anencephaly 84atrophic gastritis 83barbiturates 83birth defects 84, 86bone marrow 82, 83celiac disease 83chemotherapeutic agents 83cholestyramine 83colestipol 83colon 82Crohn's disease 83diphenylhydantoin 83DNA 82folate 79-84, see folic acidfolic acid 81-86

absorption and body stores 82Alzheimer´s disease 84atherosclerosis 84bioavailability 82blood cell formation 82cancer 83cardiovascular diseases 84chemistry 81deficiency 83dementia 84depression 84DFE (dietary folate equivalent) 84dietary sources 82disease prevention and therapeutic use 84folate coenzymes 82folate content of foods 82functions 82

97

history 86industrial production 85introduction 82measurement 82negative interactions 83neural tube defects 84positive interactions 83pregnancy 84RDA 84safety 85stability 83supplements and food fortification 85synthesis of nucleic acids 82tetrahydrofolic acid 82vitamin B12 82, 83, 84, 85vitamin B6 83vitamin C 83

homocysteine 82, 83, 84HPLC (high performance liquid chromatography) 82jejunum 82Lactobacillus casei 81, 82, 86megaloblastic anaemia 83, 84, 86methionine 82, 83methotrexate 83, 85methyltetrahydrofolate 82nonsteroidal anti-inflammatorydrugs 83oral contraceptives 83pteroylpolyglutamic acids 81pyrimethamine 83radioassays 82renal insufficiency 83RNA 82spina bifida 84, 86sprue 83sulfasalazine 83trimethoprim 83

Biotin

antibiotics 89avidin 88avidin binding assay 88biotin 87-92

absorption and body stores 88biotin content of foods 88chemistry 87coenzyme 88deficiency 89dietary sources 88disease prevention and therapeutic use 89

enteral synthesis 88functions 88groups at risk of deficiency 89history 92industrial production 91introduction 88measurement 88negative interactions 89pantothenic acid 89RDA 90safety 90stability 89supplements and food fortification 90technical applications 91UL 90

biotinidase 88, 89, 90, 92biotinidase deficiency 89birth defects 89carboxylases 88DNA replication 88energy metabolism 88fatty acids 88gluconeogenesis 88hair 88holocarboxylase synthetase deficiency 89Leiner's disease 89leucin 88nails 88seborrheic dermatitis 89vitamin H 87

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EUROPE DSM Nutritional Products Europe Ltd P.O. Box 3255 CH-4002 Basel Switzerland Phone: +41- 61-687 17 77 Fax: +41-61-688 90 22 Email: [email protected]

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