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ERN 3120 Part 1

Friday, October 9, 2015. 09:00-13:00

All questions should be attempted.

Read each question carefully before starting to answer it.

You may write your answers in either English or Norwegian. Dictionaries may be used.

There are eight questions. Each consists of two parts.

Section A requires a written answer: you should start each answer on a new page.

Section B is in the form of multiple statements, some of which are true: you should indicate which you think are correct statements by marking the box. Note: There may be more than one correct statement in each section. If you make a mistake, or change your mind, write a clear note beside the box that the mark should be ignored.

In terms of grading, all 8 questions are of equal value, and within each question parts A and B carry equal marks.

Because parts B require marking on the question sheet, you must return the question sheet marked with your candidate number, together with the written answers.

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1. Calcium A.

Describe how both dietary and internal metabolic factors influence calcium absorption in humans B. Mark the box for those statements that you agree with. One important reason for hypocalcaemia in children is:

□ Low calcium intake from food □ Low vitamin D intake □ Malabsorption of calcium □ High calcium requirement

What are the short-term consequences of calcium deficiency?

□ Hypocalcaemia □ Hyperkalaemia □ Calcification of soft tissues □ Decreased bone mass and increased risk of fractures

What are the possible long-term consequences of calcium excess from supplements?

□ Hypocalcaemia □ Hyperkalaemia □ Increased risk of myocardial infarction □ Decreased bone mass and increased risk of fractures

Which statement(s) about calcium-regulation in blood are correct?

□ PTH acts by stimulating the osteoclasts to release calcium from skeleton □ PTH acts by decreasing reabsorption of calcium in the kidney □ Calcitonin decreases calcium concentration in blood □ Vitamin D acts mainly through PTH and increases bone mass

What is the recommended calcium intake in Norway? □ Women 800 mg and men 1000 mg □ Women 1000 mg and men 800 mg □ Women and men 800 mg □ Women and men 1000 mg

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2. Vitamin A A. One of your patients, a 34-year-old female, with a history of eating disorders, is referred to you because of vitamin A deficiency. Her weight has been unchanged the last three years, and she appears healthy. Dietary assessment reveals that she eats mainly vegetables, fruits, whole grain and low-fat seafood. Vitamin A in blood is 0,50 µmol/l. Explain the main functions of vitamin A in the body, and describe the clinical symptoms that may appear in your patient if the deficiency is not corrected. B. Mark the box for those statements that you agree with. The patient should increase intake of the following food groups to improve vitamin A-status:

□ Bread and grain □ Fish and seafood □ Meat and liver □ Butter and margarine

Select the answer that is indicative of mild/moderate vitamin a deficiency:

□ Retinol < 0,1 µmol/l □ Retinol < 0,35 µmol/l □ Retinol 0,35-0,7 µmol/l □ Retinol 0,7-1,05 µmol/l

Select the retinoids that can bind to the receptors in the cell: □ All-trans retinoic acid □ 9-cis retinoic acid □ 9-cis retinal □ All-trans retinol

Which statement(s) about vitamin A metabolism are correct?

□ About 80 % of carotenoids from the diet is absorbed from the intestine □ 11-cis-retinal forms rhodopsin in the retinal cells □ Retinal is oxidized to retinoic acid in the target cells □ Vitamin A is transported by the blood as free retinol

Which dose of supplement do you recommend for patients with moderate vitamin A deficiency?

□ 500 µg Vitamin A □ 800 µg Vitamin A □ 1500 µg Vitamin A □ 3000 µg Vitamin A

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3. Water-soluble vitamins A. Which water-soluble vitamins have been linked to the occurrence of neural tube defects? Some countries have added one of these vitamins by law to all flour products; rehearse the arguments for and against this policy. B. Mark the box for those statements that you agree with. A patient with a history of alcoholism presents with several neurological signs, such as confusion, ataxia and disorientation in time or place. Which water-soluble vitamin deficiency is a possible cause? □ Folate □ B6 □ Thiamine (B1) □ Biotin Which vitamin deficiency linked to consumption of maize (corn) was associated with about 100,000 deaths in the USA in the first part of the 20th century? □ B12 (cobalamin) □ Niacin □ Pantothenate □ Riboflavin (B2) Patients undergoing bariatric surgery need to be seen by a nutritionist. Which particular vitamins (more than 1) need in particular to be monitored? □ Thiamine (B1) □ Folate □ B12 (cobalamin) □ Niacin About how much ascorbic acid is found in the typical ‘5 helpings-a-day’ of fruit and vegetables? □ 90 mg □ 500 mg □ 200 mg □ 20 mg Some water-soluble vitamins may play a role in the control of gene expression; by which mechanism(s)? □ Supply of energy in the cell nucleus □ Methylation of cytosine in DNA and of histones □ Regulation of mRNA transcription □ Demethylation of DNA and of histones

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4. Vitamin E and antioxidants A. There are many reports of short-term antioxidant intervention trials in humans, showing a decrease in DNA oxidation damage or other markers of oxidative stress. On the other hand, many clinical trials of antioxidant supplements have been carried out, with mortality or disease as endpoints. Do these two approaches give results that are consistent with each other? If not, can you suggest why this might be? B. Mark the box for those statements that you agree with. The need for vitamin E in the diet will increase with an increase in the amount of: □ protein □ saturated fatty acids □ carbohydrate □ polyunsaturated fatty acids Which vitamin(s) is/are synthesised by bacteria in the intestine? □ vitamin E □ vitamin D □ vitamin K □ vitamin A How does vitamin E act as an antioxidant? □ by acting as a free radical scavenger □ by stimulating the formation of NADPH by the pentose phosphate pathway □ by activating the formation of reduced glutathione □ by stimulating the synthesis of vitamin C The Fenton reaction □ is catalysed by heavy metals □ involves the oxidation and inactivation of a ‘suicide enzyme’ □ results in the production of a reactive free radical □ results in the inactivation of a reactive free radical Intrinsic antioxidants □ Glutathione catalyses the breakdown of hydrogen peroxide □ Glutathione (GSH), after oxidation to GSSG, is excreted from the body □ Superoxide dismutases have metal cofactors such as Cu and Zn at their active site □ Catalase contains haem (iron) at its active site

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5. Iron, minerals and trace elements A. A 55 year-old female patient has an appointment for dietary counselling at your practice. She has brought the following results from a recent blood test: Haemoglobin (Hb): 17,1 g/l, ferritin 520 µmol/l and transferrin saturation 48 %. The patient has no specific symptoms, but has felt tired for several years. She is very interested in healthy food, and uses several dietary supplements including low-dose iron, one tablet (27 mg) per day. Discuss the most likely diagnosis and at least one alternative explanation for her condition. Describe what kind of information you need before giving the patient any further advice, and why you need it. B. Mark the box for those statements that you agree with. Magnesium □ Associates with nucleotides in many enzymic reactions □ Is widely available in many different food types □ Is mostly present in the blood □ Has a structural role in the nucleus, stabilising the DNA double helix. Iodine □ Fruits and vegetables are a good source of iodine □ In Norway, feedstuffs for cattle are supplemented with iodine □ Over a quarter of the world’s population are estimated to be at risk of iodine deficiency □ Cretinism is a consequence of goitre in adults Zinc □ is stored in the liver □ Phytate is a hexose sugar, found in whole grain bread, that binds zinc and promotes its uptake in

the gut □ Zinc fingers intercalate between the base-pairs of the DNA molecule □ Zinc fingers in transcription factors are responsible for recognition of the DNA target Selenium □ can replace sulphur in the amino acids cysteine and methionine □ is a component of the antioxidant molecule glutathione □ is a component of the antioxidant enzyme glutathione peroxidase □ Fruit and vegetables are a good source of selenium Ultra-trace elements (<1 mg/day) □ Boron is an essential element in bones □ Arsenic is less toxic than, for example, selenium □ Manganese superoxide dismutase is the main antioxidant enzyme in mitochondria □ Silicon is found in connective tissue

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6. Micronutrients A. The term ‘micronutrient’ includes many phytochemicals that are not essential, but are believed to be beneficial. List the various ways in which they act in the body (excluding their possible action as antioxidants) B. Mark the box for those statements that you agree with. Flavonoids □ are efficiently taken up in the gut, and rapidly excreted □ are conjugated to aglycone carriers by microflora enzymes in the intestine □ In the Zutphen study, the major source of flavonoids was tea □ Dietary flavonoids can protect against loss of fertility in men. □ There is evidence that flavonoids inhibit the inflammatory response Carotenoids □ Phenolic groups in carotenoid molecules effectively deactivate free radicals □ Carotenoids are incorporated into lipid membranes, and so clearance from the body tends to be

slow □ Lycopene is unstable, so the concentration in canned tomato paste is lower than in fresh tomatoes □ Lutein and zeaxanthin are present in the eye, and protect the lens against UV light □ The conjugated double bonds in carotenoids render them easily oxidisable Glucosinolates and their breakdown products, isothiocyanates □ contain sulphur and account for the typical smell and taste of brassicas □ induce phase II enzyme activities, and so help to eliminate toxic compounds □ chelate transition metals and so inhibit uptake of, e.g., zinc □ inhibit the activation of certain carcinogens by phase I enzymes □ Certain glucosinolates, found in older varieties of oilseed rape, are hepatotoxic Miscellaneous □ Salicylates act as non-steroidal anti-inflammatory drugs (NSAIDs) □ Ubiquinone (Coenzyme Q) is found only in red meat □ Folate deficiency can lead to misincorporation of uracil into DNA □ Probiotics are phytochemicals that promote the growth of beneficial gut bacteria □ Folate deficiency can lead to hypomethylation of both protoconcogenes and tumour suppressor

genes, so its effect on cancer risk is hard to predict

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7. Nutrition and cancer A. What are age standardized rates (ASR), and why do we use them? Why do incidence and mortality rates of cancer not show the same geographic pattern across the world? (Some countries have very high mortality overall, but low incidence, and vice versa) B. Mark the box for those statements that you agree with. Causes of cancer □ Mutations in tumour suppressor genes tend to be seen late in the process of carcinogenesis, as

they are recessive. □ Patients with hereditary non-polyposis colon cancer have a mutation in nucleotide excision repair. □ As a factor contributing to cancer causation, family history is more important than nutrition. □ Cancer caused by endogenous damaging agents such as free radicals is known as ‘iatrogenic’. DNA damage □ Intercalating agents such as psoralens cause bridges between adjacent pyrimidines in the same

strand, that distort the double helix □ The p53 protein acts as a tumour suppressor, by directing cells with high levels of DNA damage to

apoptosis. □ Some carcinogens, such as benzo(a)pyrene, induce phase I enzymes which then deactivate them. □ ‘Signature mutations’ are found in most tumour cells, and tell us the nature of the chemical that

caused the cancer. DNA repair □ The p53 protein responds to DNA damage by delaying the cell cycle to allow repair of damage to

take place. □ Bulky adducts (e.g. aflatoxin, or benzo(a)pyrene) are repaired by mismatch repair. □ 8-oxoguanine is removed by base excision repair, leaving an apurinic/apyrimidinic (or AP-) site

which is mutagenic. □ Double strand DNA breaks are slowly repaired, and can lead to mutation, chromosome aberrations

or cell death. Cancer biomarkers □ DNA damage (e.g. 8-oxoguanine) measured in lymphocytes is a reliable indicator of cancer risk. □ Translocation of part of one chromosome to a site on another chromosome is easily seen by the

technique known as chromosome painting. □ There is a significant association between frequency of micronuclei measured in white blood cells

in healthy subjects, and later development of cancer. □ Individuals with a high DNA repair capacity are likely to be less susceptible to cancer Epidemiology □ Geographic differences in cancer incidence give important clues about the aetiology of cancer □ The main cause of liver cancer worldwide is excessive consumption of alcohol □ Lung cancer incidence is decreasing worldwide □ Incidence of breast cancer is increasing, especially in the developed world

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8. Genetic polymorphisms A.

The 'thrifty gene hypothesis' has been one of the leading hypotheses attempting to explain human obesity. Explain the concept underlying the hypothesis in a few sentences, and give arguments for and against the hypothesis.

B. Mark the box for those statements that you agree with. Polymorphisms □ SNPs can be caused by hypermethylation □ Monozygous twins are assumed to have the same SNPs □ SNPs can be caused by histone deacetylation □ Copy number variation explains most of the variation in polymorphisms. What is NOT considered to be an epigenetic mechanisms? □ DNA methylation. □ RNA associated silencing □ Histone acetylation □ DNA mutations Which observations would make you suspect that environment/lifestyle factors are important for the development of a disease? □ The symptoms appear at an old age. □ A higher frequency of the disease in close family compared to the general population. □ Different ethnic groups differ markedly with regard to disease frequency. □ Children of immigrants have a higher risk of developing the disease compared to their parents. What have we learnt from twin studies performed in the last decade? □ If monozygous twins are considerably more similar than dizygous twins for a trait, this implies that

genes play an important role in this trait. □ By studying twins from a monozygous pair who have been separated at birth, and reared apart, it is

possible to study the importance of the genetic and environmental factors for a given outcome. □ Genetic factors are important for risk of developing obesity □ Monozygous twins are epigenetically identical. Which arguments against personalised nutrition advice are valid? □ We are not yet able to give sound advice for complex lifestyle disease prevention based on genetic

information because we have limited knowledge about the gene-diet interactions. □ There are no genotypes identified that have proved to benefit from personalised nutrition. □ Advice based on gene testing is less convincing for the patient compared to advice based on

phenotype. □ Gene testing is expensive and promises too much on the ability to predict the outcome of

personalised nutrition.

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Sensor veiledning Question 1

Describe the most important factors regulating calcium absorption in humans, and how they influence the absorption.

Fasit:

Kalsiumabsorbsjonen er vanligvis mellom 10 og 30 %, og påvirkes av en rekke faktorer.

Størst betydning har vitamin D status. Når kalsiumkonsentrasjon i blodet er lav, kan absorbsjonen økes ved god vitamin D status. Dette gjelder for eksempel i sitasjoner der kroppen er i vekst og i bevegelse (med belastning på ben). Oxalat, som er en sterk hemmer med det finnes lite av i kosten. Fytat, som er en svakere hemmer enn oxalat, men det finnes mye av det I kosten.

Absorbsjonen av vanlige kalsium fra kosttilskudd avhenger av magesyre, mens tilskudd i form av kalsiumsitrat absorberes uavhengig av PH. Andre faktorer som kan redusere absorbsjonen er: mangel på magesyre, kirurgi i magesekk eller duodenum samt fettmalabsorbsjon.

Question 2 One of your patients, a 34-year-old female, with a history of eating disorders, is refereed to you because of vitamin A deficiency. Her weight has been unchanged the last tree years, and she appears healthy. Dietary assessment reveals that she eats mainly vegetables, fruits, whole grain and low-fat seafood. Vitamin A in blood is 0,50 umol/l. Explain the main functions of vitamin A in the body, and describe the clinical symptoms that may appear in your patient if the deficiency is not corrected.

Hovedfunksjonen til vitamin A I kroppen er vekst og differensiering av alle celler I kroppen, men særlig viktig for epitelceller og immunceller, samt i forbindelse med fosterutvikling. Mekanismen her er at retinsyre (all trans og 9cic) binder seg til kjernereseptorene, og påvirker uttrykk av proteiner. Vitamin A har også en viktig rolle som strukturell komponent I synsprosessen. Her fungerer 11-cic-retinal som en strukturell komponent I retina. Symptomer som kan oppstå ved vitamin A mangel er: redusert nattesyn, og eventuelt varig nedsatt syn, redusert immunforsvar (infeksjoner i luftveier og urinveier), gåsehudlignende utslett, samt at det er risiko for fosterskader dersom pasienten skulle bli gravid. Question 3

Which water-soluble vitamins have been linked to the occurrence of neural tube defects? Folate and cobalamin (B12) Some countries have added one of these vitamins by law to all flour products; rehearse the arguments for and against this policy Countries: USA, Canada and 50 others have added folic acid to flour, but none in Europe Fortification has reduced incidence of NTD pregnancies by between 20 and 50%. But the reduction depends upon the baseline prevalence. In Canadian eastern provinces the prevalence was low and fortification had only a slight effect, whereas in the western provinces there was a higher prevalence and fortification lowered the incidence by about 50% In general, in most countries, e.g. Europe including Norway the baseline prevalence is quite low at about 8-10 per 10,000 pregnancies. Below 7 per 10,000 there is no benefit from folic acid fortification (floor effect). Possibly some of the residual cases are due to low B12 status. So the arguments for folic fortification will depend upon the baseline prevalence. Even so, in most western countries to prevent one NTD pregnancy by fortification, approx..500,000 to 1 million of the population will have to be exposed to extra folic acid. Is it certain that amongst all these people no-one will suffer harm? What about cancer? The NORVIT/WENBIT trial found that those taking folic acid/B12 had an increased mortality from cancer. In USA, there was a temporary rise in cases of colorectal cancer in the 4 years after fortification was introduced. But, on the benefit side there was also a reduction in stroke mortality in both USA and Canada

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after fortification. So there is no simple answer to the question: should we fortify or not? The best policy is targeted treatment with folic acid (and B12) to those at risk, i.e. women planning to become pregnant, those at risk of stroke (e.g. those with hypertension). A recent primary prevention trial in China showed that folic acid added to an anti-hypertensive drug reduces the risk of a first stroke. Question 4 There are many reports of short-term antioxidant intervention trials in humans, showing a decrease in DNA oxidation damage or other markers of oxidative stress. On the other hand, many clinical trials of antioxidant supplements have been carried out, with mortality or disease as endpoints. Do these two approaches give results that are consistent with each other? If not, can you suggest why this might be?

Results are not consistent. While short-term trials have generally shown protective effects of antioxidants or antioxidant-rich foods, long-term clinical trials tend to show no effect or even a harmful effect, especially at high doses. The failure of clinical trials to prove the benefit of antioxidant therapies remains a great disappointment in our ability to translate our understanding of molecular and cellular function into clinical practice. Also, whereas long-term trials have been disappointing, studies of potent antioxidants in acute injury have been more encouraging (although these trials are small and yielded results that are far from conclusive). A possible explanation for the lack of benefit in clinical trials is that the trials have not lasted long enough. It may be impossible to show the benefits of antioxidant therapy over several years if the therapy is trying to reverse the results of several decades of oxidative stress. The lack of benefit seen in clinical trials to date does not disprove the central role of oxidative stress in atherosclerosis or other disease. Rather these trials challenge us to design better antioxidant trials for the future: trials that focus on using the best antioxidants at the right dose, in the right population, and for the optimal duration. Maybe the synergistic activity of combinations of phytochemicals in fruits and vegetables is effective, where a single antioxidant at relatively high dose is not. There are several possible explanations for a negative effect of antioxidant supplements on mortality. Although oxidative stress has a hypothesized role in the pathogenesis of many chronic diseases, it may be the consequence of pathological conditions. By eliminating free radicals from our organism, we may interfere with some essential defensive mechanisms such as the inflammatory response, apoptosis, phagocytosis, and detoxification. Better understanding of mechanisms and actions of antioxidants in relation to a potential disease is needed. Question 5 Den mest sannsynlige diagnose er primær hemakoroatose, på grunn av sterkt forhøyet Hb, ferritin og transferrinmetning. Alternativ diagnose er sekundær hemakromatose, på grunn av gjentatte blodtransfusjoner eller overdosering av jernpreparater over lang tid. Question 6 The term ‘micronutrient’ includes many phytochemicals that are not essential, but are believed to be beneficial. List the various ways in which they act in the body (excluding their possible action as antioxidants)

Modulation of cytochrome P450 enzymes (phase I enzymes) involved in activation of xenobiotics (prior to conjugation and excretion)

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Modulation of phase II enzymes which conjugate the reactive products of phase I to carrier molecules such as glutathione; they are then excreted

Involvement in cell signalling pathways

In the eye, protect retina from blue and actinic light

May stimulate endogenous antioxidant enzymes

Modulate DNA repair enzyme activity

Cell-cell communication (gap junctions; connexin 43 gene expression induced by carotenoids)

Taste and smell (glucosinolates) Possible action as pro-oxidants Question 8 A. In addition to the e-lecture and the references suggested during the lecture (Speakman 2004 and 2006), the students got a copy a more recent paper to provide more arguments for them to discuss the Thrifty genotype. See: Wells JCK, Disease Models & Mechanisms 5, 595-607 (2012). Some of the arguments are listed below but the students were encouraged to come up with additional arguments. I expect that the students will be able to use some of the arguments in the discussion of the thrifty genotype hypothesis. The Thrifty Genotype hypothesis (Neel, 1962) proposed that genes that are helpful under difficult circumstances become detrimental in an altered environment. Arguments for the hypothesis:

1. Human populations (ex Pimo indians of Arizona) that traditionally have gone through periodic starvation periods

• Convert more of their ingested calories into fat for storage. • Have low fat oxidation to save energy • Their diet consisted traditionally of plant food high in fibre and low in fat providing slow

glucose release. • Conversion to western diet gave high frequency of obesity and Type 2 Diabetes • 19 times higher frequency of Type 2 diabetes compared to that of US whites.

2. Many animals have strategies to save energy with regards both to use and storage. • Hibernation (dvale) • Reduced activity • Reorganization of energy-use (brain, secondary sexual features)

3. Starvation more widespread before agriculture than Speakman argues (see below). 4. Monogenic evidence on ingestion behaviour – “the greedy genotype” 5. The adipose tissue has other important functions that have been important for selection during

evolution (Elks et al., 2010a;Elks et al., 2010b; Hattersley et al., 1998, Bouchard, 2007). Some examples:

• lipid oxidation rate • appetite (Leptin) • predisposition for physical activity • Buffering starvation (adipocyte lipid storage capacity) • Buffering stochasticity (fluctations) • Buffering the brain • Reproduction and growth (Genes that are associated with adult BMI have been linked with

infant growth, pubertal timing and insulin metabolism) • Adaptation to the cold (metabolism or thermogenesis) • Immune function (secretion of cytokines) • Psychosocial stress (hirearchical ) • Sexual selection

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6. The large variation in lifestyle, physical activity etc. AND the involvement of many genes may

explain why the pattern of susceptibility genes obtained through GWAS is not clear. 7. The selection of the thrifty genes happened in a time when the selection pressure was high due to

predators/ little access to food etc.

Arguments against the hypothesis:

1. The Pimo indians changed their environment so their diet is poorer than the average white population in USA. The environmental thus may explain the higher risk of obesity in this population rather than genetic background.

2. Starvation is rarely the primary cause of death in famines, not sufficiently powerful as a source of selection to generate variability in thrifty genes (Speakman et al 2006 and 2008).

3. Starvation only after dependence on agriculture. 4. Infections are a larger cause of death (Speakman et al 2006 and 2008). 5. Speakman proposed the ‘drifty genotype’ hypothesis: millions of years through genetic drift (no

impact on phenotype) have only had impact over the last decades. 6. The list of common obesity susceptibility variants by the currently published genome-wide

association studies (GWAS) only explains a small proportion of the individual variation in risk of obesity.

7. Have developed other strategies to buffer for starvation or fluctations: Food reserves, Social networks.

8. The main difference between obese and lean people is their food intake. 9. Adaptation to cold more important for positive selection that the ability to save energy. Less

obesity in populations living in cold environments. Genes favouring «Uncoupling» (Brown Adipose Tissue) and heat production increase the chance of surviving early childhood in cold climates. Warm climate = no selection pressure due to cold adaptation.

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