Lactase Persistence and Lactase Non-Persistence

To my family and friends

Örebro Studies in Medicine 48

Ricardo Almon

Lactase Persistence and Lactase Non-Persistence Prevalence, influence on body fat, body height,

and relation to the metabolic syndrome

© Ricardo Almon, 2010

Title: Lactase Persistence and Lactase Non-Persistence. Prevalence, influence on body fat, body height,

and relation to the metabolic syndrome.

Publisher: Örebro University 2010www.publications.oru.se

trycksaker@oru.se

Printer: Intellecta Infolog, Kållered 11/2010

issn 1652-4063 isbn 978-91-7668-767-3

3

Abstract

A single nucleotide polymorphism (SNP) in chromosome 2 gives humans the capability to continue digesting lactose after weaning. Among individuals of European descent it is the SNP (LCT-13910 C>T), which is located 14 kb upstream from the start of transcription of the lactase gene (LCT). A C to T mutation enables the continued production of the enzyme lactase throughout life, and thereby the digestion of lactose throughout life without symptoms of lactose intolerance. The trait is called lactase persistence (LP). LP is an autosomal dominant trait. In contrast, lactase non-persistent (LNP) individuals show a decline of lactase production after weaning. LNP individuals habitually show symptoms of lactose intolerance after consumption of milk and some milk products. Using the LCT -13910 C>T SNP we reassessed the prevalence of LP/LNP in Sweden. We increased the accuracy of our estimates by using Hardy-Weinberg’s formula for allelic frequencies. We found a prevalence of 14% for LNP. This is about 5.5 fold higher than the prevalence formerly assumed for Sweden (range: 1-5%). Childhood milk consumption has become normative since the beginnings of the last century. Studies using milk supplements in schools, performed in the UK in the 1920s, showed that childhood milk consumption led to gains in body weight and height in children. We readdressed this question given the changed socioeconomic settings a century later. In today’s, nutritionally replete socioeconomic settings childhood obesity is, instead of stunting and undernourishment, the target of public health nutrition. We did not find evidence for higher measures of body fat coupled to consumption of milk and milk products or the lactase genotype in Swedish children and adolescents. We found, nevertheless, a positive association between milk intake as well as LP and body height in Swedish children and adolescents. Finally, we studied if LP/LNP, using Mendelian randomization (MR), affects the development of the metabolic syndrome in adults. We chose an accessible population from the Canary Islands in Spain with a prevalence of 40% for LNP and 60% for LP. We found a positive association of LP with metabolic syndrome. LP subjects of the Canary Islands exhibited a 57% higher risk to develop metabolic syndrome compared to LNP subjects. Interestingly, LP women showed a 93% higher risk to develop MS compared to a 22% higher risk in LP men. In conclusion, the previously not known relatively high prevalence of LNP for Sweden impacts health care and public health policy decisions. Childhood milk consumption affects longitudinal growth, but not body fat mass in nutritionally replete countries like Sweden. The long-term effects of childhood milk consumption need to be elucidated. Non-caloric milk constituents might influence the somatotropic axis of children, which can have both positive and negative long-term effects. LP status and milk consumption may increase the susceptibility to develop metabolic syndrome in some populations. Non-caloric constituents of milk might exert sex specific effects increasing the risk to develop metabolic syndrome in susceptible populations.

Keywords: LCT -13910 C>T polymorphism, prevalence, lactase persistence and lactase non-persistence, milk and milk product intake, body fat, body height, metabolic syndrome Ricardo Almon, Family Medicine Research Centre, School of Health and Medical Sciences, Örebro University, Box 1613, 70116 Örebro, Sweden. E-mail: ricardo.almon@orebroll.se

4

SammanfattningOrsaken till att vissa människor kan metabolisera mjölksocker efter avvänjning är en genmutation (single nucleotide polymorphism (SNP)) i kromosom 2. Bland européer lokaliseras SNP (LCT-13910 C>T) 14 kb uppströms från starten av transkription av laktasgen (enzymet som bryter ned mjölksocker kallas laktas). Mutationen ger en livslångd bibehållen laktasproduktion. Tillståndet kallas laktaspersistens (LP). Däremot kallas tillståndet, när man tappat förmågan att producera laktas efter avvänjning, laktas non-persistens (LNP). Individer som är LNP får symtom efter konsumtion av mjölk och vissa mjölkprodukter och detta kallas för laktosintolerans. Med hjälp av LCT-13910 C>T SNP skaffade vi nya estimat för LP/LNP prevalensen i Sverige. Vi höjde precisionen av estimat genom att använda oss av Hardy-Weinberg’s ekvation för allelfrekvenser. Vi hittade en prevalens på 14 % för LNP. Detta är ungefär 5,5 gånger högre än traditionella prevalensdata för Sverige (variationsvidd: 1-5 %). Mjölkkonsumtion i barndomen har blivit normativ sedan begynnelsen av senaste seklet. Studier bland skolbarn i Storbritannien visade att de barn som fick mjölktillägg till skolmaten blev längre och ökade i kroppsvikt. I dagens nuläge är problemet barnobesitas i folkshälsofokus och inte växtretardering eller malnutrition. Vi hittade ingen effekt av mjölkintaget på kroppsfetma eller vikt hos barn. Däremot hittade vi en positiv association mellan mjölkintag och kroppslängden hos svenska barn och ungdomar. LP statusen visade också en positiv association till kroppslängden. Vi undersökte dessutom i en representativ vuxenpopulation från Kanarieöarna i Spanien om LP/LNP uppvisar en koppling till metabola syndrom. Populationen uppvisade en frekvens på 40 % på LNP och 60 % på LP. Vi hittade en positiv association mellan LP och metabola syndromet. LP individer på Kanarieöarna visade en 57 % högre risk att utveckla metabola syndromet jämfört med LNP-individer. LP kvinnor visade en 93 % högre risk att utveckla MS jämfört med 22 % högre risk för LP män. Sammanfattningsvis visades att prevalensen 14% för LNP i Sverige är mycket högre än de traditionella siffrorna. Resultaten har en folkhälsorelaterad betydelse men också en klinisk betydelse. Mjölkkonsumtionen i barndomen verkar ha en effekt på kroppslängden hos barn. Ingen effekt av mjölkintaget på vikt eller kroppsfetma hittades. Långtidseffekterna av mjölkintag fortsätter att vara oklara och debatteras kontroversiellt. Sannolikt har icke kaloriska beståndsdelar i mjölken en effekt på kroppslängdsutveckling hos barn. Dessa kan ha både negativa och positiva konsekvenser. LP-statusen bland vuxna på Kanarieöarna kan eventuellt öka genetiska mottagligheten för att utveckla MS. Icke kaloriska element i mjölk kunde möjligtvis ha könsspecifika effekter i vissa populationer som ökar risken att utveckla metabolt syndrom.

5

List of publications

1. Almon R, Engfeldt P, Tysk C, Sjöström M, Nilsson TK. Prevalence and trends in adult-

type hypolactasia in different age cohorts in Central Sweden diagnosed by genotyping for the

adult-type hypolactasia-linked LCT -13910C > T mutation. Scand J Gastroenterol.

2007 Feb;42:165-70.

2. Almon R, Patterson E, Nilsson TK, Engfeldt P, Sjöström M. Body fat and dairy product

intake in lactase persistent and non-persistent children and adolescents. Food Nutr, Res;

2010; 54.

3. Almon R, Nilsson TK, Sjöström M, Engfeldt P. Milk consumption and body height in

preadolescent and adolescent children. (submitted) 2010.

4. Almon R, Alvarez-Leon EE, Engfeldt P, Serra-Majem L, Magnuson A, Nilsson TK.

Associations between lactase persistence and the metabolic syndrome in a cross-sectional

study in the Canary Islands. Eur J Nutr. 2010; 49:141-146

6

List of abbreviations

AtH Adult type Hypolactasia

ATP III Adult Treatment Panel III

BMI body mass index

CVD cardio vascular disease

EYHS European Young Heart Study

ENCA Canarian Nutrition Survey

IDF International Diabetes Federation

IGF-I insulin-like growth factor I

LCT lactase

LNP lactase non persistence

LP lactase persistence

LPH lactase phlorizin hydrolase

MCM6 mini-chromosome maintenance gene

MR Mendelian randomization

MS metabolic syndrome

NCEP National Cholesterol Education Program

7

1. Introduction

1.1. Lactase persistence (LP) and Lactase non persistence (LNP) ________________ 8

1.1.1. Prevalence and genetics of LP/LNP _____________________________________ 8

1.1.2. Detection of LNP ___________________________________________________ 11

1.1.3. Milk and milk product consumption across Europe _________________________ 12

1.1. 4. Milk intake, body composition and growth among children and adolescents _____ 14

1.1. 5. Milk intake and the Metabolic Syndrome ________________________________ 16

2. Aims ______________________________________________________________ 18

2.1. Study 1 ____________________________________________________________ 18

2.2. Study 2 ____________________________________________________________ 18

2.3. Study 3 ____________________________________________________________ 18

2.4. Study 4 ____________________________________________________________ 18

3. Participants, Materials and Methods ___________________________________ 19

3.1. Participants ________________________________________________________ 19

3.1.1. Swedish Participation - European Youth Heart Study (EYHS) _________________ 19

3.1.2. Active Seniors ______________________________________________________ 20

3.1.3. Spanish Participation - Nutrition Survey of the Canary Island (ENCA) __________ 21

8

3.2. Materials and Methods _______________________________________________ 22

3.2.1. Material and Methods (EYHS) __________________________________________ 22

3.2.2. Material and Methods (NSCI/ENCA) _____________________________________ 23

3.2.3. Genomic Analysis ____________________________________________________ 24

4. Methodological Consideration ________________________________________ 25

4.1. Hardy-Weinberg-Equilibrium (HWE) ____________________________________ 25

4.2. Mendelian Randomization (MR) ________________________________________ 26

5. Main Results and Discussion _________________________________________ 27

5.1. Study 1 ____________________________________________________________ 27

5.2. Study 2 ____________________________________________________________ 28

5.3. Study 3 ____________________________________________________________ 29

5.4. Study 4 ____________________________________________________________ 31

6. General Discussion and future Perspectives _____________________________ 32

8. Acknowledgements __________________________________________________ 36

9. References _________________________________________________________ 37

11

9

1. Introduction

1.1. Lactase persistence (LP) and non-persistence (LNP) 1.1.1. Prevalence and Genetics

The consumption of milk and milk products has long traditions in human nutrition.

Hippocrates first described lactose intolerance around 400 years BC.1. Differences in the

ability of human adults to digest milk have also been remarked upon in Roman times. Milk

and dairy products contain lactose, the sugar component of almost all milk. The disaccharide,

lactose, is a unique carbohydrate present only in mammalian milk. Lactose is hydrolyzed to

glucose and galactose in the brush border of the small intestine by the enzyme lactase

phlorizin hydrolase (LPH), more commonly known as lactase (LCT). Lactase is essential for

the nourishment of newborn mammals, whose sole source of nutrition is milk, in which

lactose is the major carbohydrate component. In adult mammals other than in humans lactase

production decreases significantly in quantity following weaning. The maturational decline in

lactase activity renders most of the world’s adult population intolerant of excessive

consumption of milk and other dairy products containing lactose. About 30% of the world

population has continued lactase activity, i.e. is LP, beyond weaning and into adulthood 2.

LP appeared in Eurasia about 10,000 years ago. Evidence for LP in Europeans, prior to the

Neolithic, has not been found yet3. Fully developed dairying-based farming economy emerges

first during the late Neolithic in Southeast Europe and the Middle Neolithic cultures following

the Linearbandkeramik in Central Europe and is mainly connected to cattle in Central Europe.

The spread of the Neolithic lifestyle from Aegean to Central Europe goes hand in hand with

the decline of the importance of sheep and goat and the rise in frequency of cattle bones in

archaeological assemblages. Historic reports and archaeological findings point that in

Southern Europe goat and sheep, and to lesser extent cattle, based economies used processed

milk instead of fresh milk. The Romans, for example, used goat and sheep milk for the

production of fermented milk products and cattle mainly as draught animal. In contrast,

Germanic peoples and other habitants of Central and Northern Europe practiced cattle

dairying and drank significant amounts of fresh milk. The Romans, on the other hand,

12

10

considered the consumption of fresh milk as barbaric and simple, instead processing it to

yoghurt, cheese and other milk derived products.

The appearance of LP is most probably the result of a gene-culture co-evolution in contrast to,

for example Malaria-associated haemoglobinopathies, which can be regarded as results of

gene-environment interactions. Concerning LP, culture, economy and population history

appear to be the driving force that led to the expansion of LP, and the frequencies of LP we

observe today in Europe might be the result of a recent and strong natural selection 4-9. While

not fully understood, the biological advantages of LP might include the continuous

availability of an energy rich drink that enables farming communities to overcome poor

harvests. Also, a combination of both theories is discussed as the driving force behind the

expansion of LP. It also is possible that there exist additional unidentified selective

advantages of milk consumption, which could explain natural selection.

Variation in expression of lactase was not established as genetically determined trait until the

second half of the twentieth century. Before this, LP was considered by people of European

descent to be the physiologically “normal” state, and widespread LNP in adults was only

appreciated in the early 1960s 10-11. By the early 1970s it was established that the LP

polymorphism in humans had a genetic cause, and is inherited in an autosomal dominant

manner 12. In the 1980s, Ho et al reported a trimodal distribution of LCT. The trimodal

distribution was interpreted as attributable to groups of individuals homozygous for LP

(highest lactase activity), heterozygous for LP (mid-level activity) and LNP (low lactase

activity) 13.

LP is inherited as dominant Mendelian trait in Europeans 14-15. This trait shows a high

frequency in Northern Europeans (>90% in Swedes and Danes) and is also common in

pastoralist populations in East-Africa (~90% in Tutsi, ~50% in Fulani). It decreases in

frequency across Europe (~50% in Spanish, French and pastoralist Arab populations) and is

low in non-pastoralist Asian and African populations (~1% in Chinese, ~5% - 20% in West

African agriculturalists) 15.

The decline in lactase expression is usually complete during childhood, but the decline has

also been reported to occur later in adolescence 16. The rate in which lactase activity declines

can vary according to ethnicity. The reasons for this difference in timing are not known.

13

11

Postdecline, the level of lactase activity is 5-10% of childhood levels in most populations

worldwide 17. In Northern Europeans it may take up to 18-20 years for lactase activity to

reach lowest expression 1. However, some individuals, particularly descendents from

populations that have traditionally practiced cattle domestication, maintain the ability to

digest milk and other dairy products into adulthood 18.

A single nucleotide polymorphism (SNP) C>T -13910 located 14 kb upstream of the lactase

gene (LCT) in the MCM6 (mini-chromosome-maintenance6), gene (Figure 1) has been shown

to be associated with LP (LCT-13910 T allele) in individuals of European descent, but varies

widely in frequency across Europe 4 (Figure 2). The allele appears to be dominant over the

LCT-13910 C allele that represents LNP. Accumulating data imply the critical functional role

of the LCT-13910 region 19. However, there are recent reports of other mutations at, or near

this site: -13907, -13915, -13913, -13914, and -14010 variants that correlate with LP in

different populations 18, 20. Furthermore, to test for LP status for people of East African or

Arabian ancestry using only LCT-13910 C>T can be considered inappropriate 21. Data

indicates the lack of the LCT -13910 T variant in most Sub-Saharan populations, known to

show high prevalence of LP, implying thus that other LP mutations must exist globally.

Recently this has been confirmed in Chinese populations. A variant at the locus LCT -22018

reflected LP in 6 different populations and the authors suggested that, taking together the

results of previous studies, the origins of LP associated alleles are different in different

pastoral populations 22. LP shows thus independent origins in varying populations worldwide

which would imply thus convergent evolution.

Homozygosity for the C allele (LCT-13910 CC) shows for all practical purposes a complete

association with LNP in most populations of European origin 14, 23-25. Homozygosty for the C

allele is a contributing factor associated with the reduction of milk and some dairy products

consumption to approximately 12 g lactose or two cups of milk daily in adults and some

children and adolescents 26. Typical symptoms of higher consumption of these products can

include pain, cramps, bloating, flatus, diarrhea and borborygmi 1, 27. Molecular epidemiologic

studies have shown that the prevalence of LNP is consistent with previously published

phenotypically determined epidemiologic data on LNP in more than 70 countries 28. The

majority of heterozygotes, having intermediate levels of lactase activity in intestinal biopsies 29 are traditionally thought to produce sufficient lactase to be classified by the standard

physiological tests as lactose digesters 30.

14

12

Figure 1: The physical map showing the analyzed genome region flanking the C/T-13910

associated with LP. The distance (in kb) from the first ATG of LCT is shown. A, Genes in the

region studied. B, Expanded map of the 30-kb region in the LCT and MCM6 genes, showing

SNPs 1–9 analyzed in the population samples.20 (Courtesy of Enattah et al.)

1.1.2. Detection of LP and LNP

Genotyping for LNP and LP detects primary lactase deficiency (i.e. LNP) and the genetically

determined unrestricted capacity to digest lactose even in adulthood (i.e. LP). LNP can but not

always have to correlate with lactose maldigestion, and lactose maldigestion can, but not

always have to correlate with symptoms of ‘lactose intolerance’.

The term ‘lactose intolerance’ is commonly used to indicate symptoms experienced after

consumption of milk or milk products. The symptoms can be mild, moderate or severe. The

expression of symptoms depends on a number of factors. These factors include: quantity of

consumed milk, fat content of the milk, rate of stomach emptying, oro-cecal transit time,

individual sensitivity to abdominal discomfort, capacity of bacteria in the colon to digest

lactose not absorbed in the small intestine, psychological impact of anticipation of symptoms

in those who have had previous symptoms during drinking milk (nocebo-effect).

The detection of lactose maldigestion can usually be made on the basis of the history,

supported by dietary manipulation 31. Diagnostic tests range from biochemical (low fecal pH

indicates lactose maldigestion used primarily in infants) over functional tests (e.g. hydrogen

15

13

breath test or serum glucose levels after ingestion of standard doses of lactose) to biopsy of

the small bowel and direct assay of intestinal enzymatic activity.

Especially in children, genotyping for LNP/LP to detect ‘lactose intolerance’ appears

inappropriate, also on account of the important differences in timing of decline of lactase

expression in these age groups 1, 16. In this age group a good clinical history often reveals a

relationship between ingestion of lactose and symptoms. When lactose maldigestion is

suspected a lactose-free diet can be tried. It is important that all sources of lactose be

eliminated, requiring the reading of food labels to identify ‘hidden’ sources of lactose.

Generally, a 2-week trial of a strict lactose free diet with resolution of symptoms can be

diagnostic. In more subtle cases, the hydrogen breath test is the least invasive and most

helpful test to detect LNP 32.

Genotyping for LNP/LP can be used as a screening test in adults 28. Combinations of this

technique with functional tests, including not only hydrogen but also methane measurement

have been described 33. Nevertheless, whatever result is gained by whatsoever technique, if

lactose consumption are thought to produce symptoms, diagnosis must be confirmed by

disappearance of symptoms following a lactose free diet. Thus, a major disadvantage of

genotyping for LNP/LP is that it obviously does not indicate if a subject has or does not have

symptoms of lactose maldigestion.

The advantages of genotyping for LNP/LP are that the test is non-invasive and that it saves

both time and cost in healthcare. These circumstances, on the other hand, make genotyping

for LNP/LP also to suitable test for large-scale screenings for LNP/LP in different populations

or for studies with epidemiologic purpose.

1.1.3. Milk and milk product consumption across Europe

The present work studies two populations: one representative, randomly sampled population

of 694 healthy children and adolescents belonging to Swedish section of the European Youth

Heart Study (EYHS) and a representative sample of 551 healthy adults from the Canarian

general population randomly sampled in the Canarian Nutrition Survey (ENCA). Further

details about these studies can be found in the section Material and Methods. Between these

16

14

populations there exists a significant difference of the prevalence of LP/LNP. For Sweden the

figures of prevalence of LP are one of the highest in the world with about 90%. In contrast,

the prevalence of LP in Spain is about 50% 15.

Cow’s milk consumption is highest in Finland with 184 liters (l) per capita and year followed

by Sweden with 146 l per capita and year. Ireland with 130 l and Netherlands with 123 l per

capita and year are also among the countries with highest milk consumption. Spain,

interestingly, with a consumption of 119 l milk per capita and year shows higher figures than

countries like Norway (117 l), the United Kingdom (UK) (111 l), Germany (92 l) and

Switzerland (113 l) (Source: International Dairy Federation, Bulletin 423/2007). The

European Prospective Investigation into Cancer and Nutrition Study (EPIC), showed different

dietary patterns among Europeans. The authors suggested two major dietary profiles among

Europeans. The northern profile is characterized by low consumption of fruit and

pasta/rice/other grain, vegetables, bread and wine, fish, legumes and vegetable oils, and high

in consumption of butter, potatoes, dairy products and other cereals, such as flour, pastry and

breakfast cereals, meat, non-alcoholic and alcoholic beverages except wine, sugar and

cakes 34.

Based on the EPIC study, Hjärtåker et al could identify different patterns of milk and dairy

consumption across Europe 35. The Spanish and Nordic countries reported a high consumption

of milk; the Swedish (particularly Umeå) and but also Dutch centres reported high

consumption of yoghurt and other fermented milk products, whereas the highest consumption

of cheese is reported in French centers. The UK general population group and the Dutch

centers showed the highest consumption of cream desserts and milk-based puddings. Italian

and Nordic centers showed the highest consumption of ice-cream. As for butter, Germany, the

UK and French reported highest intakes. The studied data interestingly suggested that

countries consuming lower quantities of milk (e.g. Greece and Germany) tend to consume

higher quantities of cheese 35.

17

15

Figure 2: Interpolated map of the ‘old world’ showing the distribution of Lactase persistence;

data taken from the literature 36. (Courtesy of Ingram et al)

1.1.4. Milk intake, body composition and growth among children and adolescents

Female mammals produce milk for the sole purpose to feed their offspring, a fact that lead us

to hypothesize that milk might have some unique biological effects. The inclusion of milk and

dairy from another mammalian order in human diets is evolutionary novel, compared to the

time since mammals exist and to hominid evolution. But, humans are also the only species to

wear skins of other animals and the only species to cook our food. These circumstances point

to the adaptability of humans and are not always to inappropriate behavior. Nevertheless, milk

is composed of an array of bioactive molecules that shape the development of nursing infants,

who accrue body size (weight and linear dimensions) while organ systems mature.

Specifically calves grow rapidly in skeletal size and weight, gaining in average 0.7-0.8 kg per

day in the first year of life. In contrast, breastfeed infants gain in average about 0.02 kg per

day in the first year of life (WHO, 2007). Cow’s milk supports thus biologically a much faster

rate of increase in body size. Furthermore, formula-fed infants show greater gains in weight

and length compared to breastfeed infants 37. The question we study in this work is, whether

18

16

routine consumption of cow’s milk generates gains in weight in children and adults, and in

body height in adolescent and preadolescent children.

Human height is a highly complex trait. Within a population about 80-90% of the variation in

body height is due to genetic factors 38. In modern western societies, about 10-20% of the

variation in body height is due to environmental factors. The most important environmental

factors affecting longitudinal growth are nutrition and diseases 39.

Milk consumption is one of the most studied nutritional factors in relation to height and

growth rate in humans. Already in the early 1900s very well performed interventional studies,

offering milk supplements to schoolchildren, showed that cow’s milk led to an increased

longitudinal growth 40-41.

Today, almost a century later, the long-term effects of childhood milk consumption on growth

is still debated. There is, however, considerable evidence that cow’s milk stimulates

longitudinal growth 42-43. Non-caloric, bioactive growth-promoting constituents, which

influence the insulin-like growth factor I (IGF-I) – axis leading to higher circulating levels of

IGF-I, are considered to be responsible for the growth stimulating effect of cow’s milk in

nutritionally replete, industrialized countries 44-50.

Milk and other dairy products are currently the primary source of calcium in western diets and

milk avoidance has been linked to small stature 51-52. Swedish children and adolescents were

of specific interest for our analysis of potential effects of milk consumption and longitudinal

growth due to the high prevalence of lactase persistence (LP) 15, 53, and on account of the

presence of one of the highest rates of milk consumption per capita in Europe and in the

world 35.

As regards our study on potential effects of milk intake on body height in preadolescent and

adolescent children we noticed with surprise that in similar studies no adjustments for parental

height had been performed despite the large body of publications. We were able to adjust our

statistical models, among other possible confounders, for birth weight and parental height,

which are important covariates in relation to subsequent growth (as discussed

above) 54.

19

17

1.1.5. Milk intake and the metabolic syndrome

The concept of the metabolic syndrome is a cluster of interrelated risk factors for

cardiovascular disease (CVD) and type 2 diabetes mellitus. The factors include dysglycemia,

raised blood pressure, elevated triglyceride (TG) levels, low high-density lipoprotein

cholesterol (HDL) levels and obesity (particularly abdominal obesity). Several metabolic

syndrome definitions exist 55-58. Clinical definitions of the metabolic syndrome vary also in

the priority given to obesity. The International Diabetes Federation (IDF) made abdominal

obesity as necessary requirement in the diagnosis of the metabolic syndrome 59. We have used

the IDF definition of the metabolic syndrome in the present work. We consider obesity being

central to the metabolic syndrome. Obesity is important years before the development of the

other disorders associated to the metabolic syndrome become manifest. We believe that

obesity, even seen separated, can be considered as an important risk factor for future

metabolic derangement. The IDF definition of the metabolic syndrome is defined by central

obesity (wait circumference in men ≥ 94 cm and in women ≥ 80 cm plus two of the following

four criteria: fasting glucose ≥ 100mg/dl, TG ≥ 150 mg/dl (or pharmacological treatment),

HDL cholesterol in men < 40 mg/dl, in women < 50 mg/dl (or pharmacological treatment),

systolic blood pressure > 130 mmHg and diastolic blood pressure > 85 mmHg (or

pharmacological treatment).

The metabolic syndrome appears almost as a ‘lifestyle disorder’. Otherwise, therapeutic

lifestyle changes, such as ‘healthy eating patterns’ and increased physical activity, would not

show the positive impact on the metabolic syndrome they overwhelmingly show. Overeating

and sedentary lifestyles play an important role in the development of the metabolic syndrome.

Also defined dietary patterns might influence the development of the metabolic syndrome in

genetically susceptible subjects. The observation that elements of the diet beyond the caloric

content can have an impact on the metabolic syndrome has led many investigators to question

which specific food elements may influence the metabolic syndrome.

Research also indicates a substantial role for genetic susceptibility to the metabolic syndrome 60. There are a growing number of studies investigating potential gene-nutrient interactions.

Given that by definition the metabolic syndrome is coupled to different criteria, the analysis

of potential gene-environment and gene-nutrient interactions is complex. To disentangle the

relation of individual genes on the metabolic syndrome in the presence of multiple putative

20

18

SNPs and highly variable environmental factors may help to improve efficacy of dietary and

other lifestyle recommendations. These circumstances led us to investigate if the LCT-13910

C>T polymorphism has an effect on the metabolic syndrome.

Due to the complexity of the studied matter we introduced the concept of Mendelian

randomization (MR). By subdividing genotypes in LP and LNP we readdress the question of

whether nutrigenetically defined factors, like milk and milk product intake, affect the

metabolic syndrome. MR studies modifiable causes of disease in genetic epidemiology. A

functional genetic variant, in our study LCT-13910 C>T polymorphism, acts as proxy for

modifiable lifetime exposure patterns, i.e. milk consumption. The LCT-13910 C>T

polymorphism is known to influence the exposure pattern, milk consumption 20, 26, 61-64. MR is

described in more detail in section 4.2 (methodological considerations) below.

Some studies show that moderate increase of milk and milk product consumption have

protective effects against the metabolic syndrome 65-67, high blood pressure 68-69, and influence

positively unfavorable LDL and HDL profiles 70. Other studies suggest that milk and milk

product consumption may help prevent weight gain and even promote weight loss 66, 71. Other

studies in turn suggest that milk avoidance is associated with a reduced risk of MS 72 and in

children there have been found a positive association between milk consumption and higher

body mass indexes (BMI) 73-74. The role of milk consumption as regards MS and obesity is

thus still controversially debated.

We chose for the present study concerning possible effects of milk intake on MS an available

population of the Canary Islands in Spain, which we genotyped for LCT-13910 C>T

polymorphism. The Canary Islands were of special interest because in contrast to the rest of

Spain, there is a high prevalence of cardiovascular risk factors and one of the highest

cardiovascular mortality rates of the country, particularly among women. In addition, the

Canary Islands display one of the highest rates of milk consumption in Spain, comparable or

even higher than in some Nordic countries

21

19

2. Aims

2.1. Study 1: to determine the prevalence of LNP/LP in Sweden by genotyping for the

LCT-13910 C>T SNP.

2.2. Study 2: to assess if LNP/LP, defined by genotyping for the LCT-13910 C>T SNP, is

associated with differences in milk and milk product intake, and to evaluate if milk and milk

product consumption is related to differences in measures of body fat in children and

adolescents.

2.3. Study 3: to assess if milk intake and LNP/LP, defined by genotyping for the LCT-13910

C>T SNP, is associated to differences in body height in preadolescent and adolescent

children.

2.4. Study 4: to assess if LNP/LP, defined by genotyping for the LCT-13910 C>T SNP,

is related to the metabolic syndrome in the Canary Islands in Spain.

22

20

3. Participants, Material and Methods

3.1. Participants 3.1.1. The European Youth Heart Study (EYHS)

The EYHS is a school-based, cross-sectional study designed to examine the interactions

between personal, environmental and lifestyle influences on risk factors for future CVD. A

scientific Committee of senior researchers designed EYHS. Core original members of the

committee were Drs. Chris Riddoch (UK), Michael Sjöström (Sweden), Karsten Froberg

(Denmark) and Dawn Edwards (UK). EYHS data have been collected in five countries:

Denmark (Odense, 1997/1998), Sweden (Örebro and Stockholm, 1998/1999), Estonia (Tartu,

1998/1999), Portugal (Madeira, 1998/1999) and Norway (Oslo, 1999/2000). All participating

EYHS countries followed the protocol manual, and to attain comparable results between the

countries meetings were held for new groups and one central laboratory was used for serum

analysis 75-76.

The present work has been developed with data obtained from the Swedish section of the

European Youth Heart Study (EYHS). The Swedish part of the study began with cross-

sectional data collection in 1998-1999 (EYHS-I). Repeated observations of the key indicators

addressing cardiovascular risk factors and their determinants were performed in 2004 - 2005

(EYHS-II). The present work is based on EYHS-I and thus cross-sectional data. The sampling

frame in Sweden included the municipality of Örebro and the southern region of Stockholm.

The municipality of Örebro had 160,000 inhabitants, of whom there were about 1,500 pupils

in each of the EYHS age group (9 and 15 years). The south region of Stockholm, defined here

by the municipalities of Botkyrka, Haninge, Huddinge, Nynäshamn, Salem, Södertalje and

Tyresö, had about 370,000 inhabitants, of whom there were about 5,000 pupils in each of the

two age groups.

A two-stage cluster procedure was employed in each of the study locations. In the first step all

public schools within each local area were identified and stratified by target age groups and

the mean income level (below or above the mean of their municipality) of the respective area.

Subsequently, schools were randomly sampled within each stratified age group (9 and 15

years) and invited to partake. In a second step, groups of children and adolescents of the

23

21

predefined target age were randomly sampled from each school in proportion to school size

and then invited to participate. The aim was to obtain 250 participants in each age or gender

group (totally 1,000 participants). Fifty per cent oversampling was performed on the basis of

experiences from a pilot study 77. The overall participation rate in Sweden was 50%. In total

1154 subjects agreed to participate in the EYHS. Twenty four per cent declined and 26 % did

not respond at all. Of those to agreed to partake in the study 17 (1%) failed to keep their

appointment rending finally 1137 participants. A comprehensive analysis of the non-

participants was carried out including questionnaire to be filled out by the schoolchildren. The

most common reason given for non-participation was ‘I didn’t want to have a blood sample

taken’; 38 % of the preadolescent and 33 % of the adolescent children gave this answer.

Participation in this study was quite demanding (eg blood sampling and maximal ergometer

bicycle test). The socioeconomic background of participants was slightly above average

compared to the general Swedish population. Study design, sampling procedure, participation

rates and study protocol have been reported in detail elsewhere 78. We used in the present

study a sub-set of 684 schoolchildren as they had been profiled for the LCT-19310 C>T

polymorphism.

3.1.2. Active Seniors

The second study population used to explore potential secular trends in the prevalence of

LP/LNP in Sweden consisted of 392 elderly individuals (Active Seniors). This cohort

constituted a non-randomly sampled cohort recruited from study circles for the elderly. The

locations for the recruitment were selected to represent a broad range of socioeconomic levels

and included rural as well as urban and sub-urban areas. Being retired (usual age ≥ 65 years in

Sweden) and living independently in their own homes in addition to the ability to participate

in the study circles, were the sole inclusion criteria, not preset health criteria. However, this

cohort of elderly subjects was not primarily recruited to partake in the present study. All were

of European descent. The majority of the subjects of this study population were born between

1920 and 1932; the mean year of birth was 1928.

24

22

3.1.3. Nutrition Survey of the Canary Islands (ENCA)

The Canary Islands are a Spanish region formed by seven islands located in the Atlantic

Ocean off the African coast of Morocco. The Canary Islands are a Spanish autonomous

community and has about 2 million inhabitants and enjoy a sub-tropical climate. The name of

the archipelago is probably derived from the latin, Insula Canaria, meaning “Island of the

Dogs”. The original inhabitants were the “Guanches”; they worshiped dogs as holy animals.

Also the ancient Greeks knew about these islands far to the west, whose habitants were the

“dog-headed ones”. Phoenicians and Carthaginians have visited the islands. The history of the

settlement of the archipelago is still unclear. Linguistic and genetic analyses point to possible

common origin with the Berbers of northern Africa. When Europeans began to explore the

islands they found several indigenous populations living at a Neolithic level of technology. In

1402 the Castilians began to conquer the archipelago, with the expedition of French

adventurers, Jean de Béthencourt and Gadifer de la Salle, nobles and vassals of Henry III of

Castile. Complete pacification of the resistance of the native Guanchos was achieved 1495.

Thereafter the Canary Islands were incorporated in the Kingdom of Castile.

Today, and since establishment of a democratic constitutional monarchy after the end of

Franco’s regime in 1975, important socioeconomic changes have had an impact on food

consumption and health status. In contrast to what is found in the rest of Spain, in the Canary

Islands a high prevalence of cardiovascular risk factors exists together with one of the highest

cardiovascular mortality rates of the country, especially among women. ENCA showed

several particular features for the population of the Canary Islands. The Canary Islands

display on account of their history and geographic location nutritional singularities when

compared to the rest of Spain. ENCA revealed an ongoing nutritional shift from a

Mesoamerican to a more Mediterranean pattern of food consumption, nevertheless

maintaining their sub-tropical Canarian characteristics. Furthermore, the “Tablas de

Composición de Alimentos Españoles” (tables of composition of Spanish food items) of

Mataix et al 79 used in the nutritional survey were revised and 55 food items typical for the

Canary Island required to be added. In addition, the tables of composition of Spanish food

items were completed by French tables of composition of food items 80.

25

23

In the Canarian Nutrition Survey (ENCA 1997-1998) a representative sample of the Canarian

general population between 6- and 75-year-old. A total of 1747 individuals participated.

Analysis of non-participation was performed. Details on data collection and methodology

have been published in detail elsewhere 81-84.

3.2. Material and Methods

3.2.1. Material and Methods EYHS

Blood samples were obtained from 684 children (334 girls and 350 boys) belonging to the

Swedish part of the European Youth Heart Study (EYHS). Height, weight, hip and waist

circumferences, as well as skinfold thickness were measured by standardized procedures 85.

Body mass index (BMI) was calculated as weight/height² (kg/m²). Body fat percentage was

calculated from the equation by Slaughter, based on skinfold thickness measurements 86. The

skinfold measures have been shown to correlate highly with dual-energy X-ray

absorptiometry-measured body fat percentages in children of similar ages 87. The skinfold

measurements were taken using a Harpenden caliper at the biceps, triceps, subscapular,

suprailiac and triceps surae sites on the left side of the body. All measurements were taken

twice and the mean calculated. If the difference between the 2 measurements differed

by > 2 mm, a third measurement was taken and the 2 closest measurements were averaged.

The consumption of milk and milk products was assessed by an interviewer-mediated 24-hour

recall. A qualitative food record completed the day before the interview with the help of

parents served as a checklist for the data obtained during the recall in the nine-year-olds. A

food atlas was used to estimate portion sizes. Dietary data were processed by StorMats

(version 4.02, Rudans Lättdata, Sweden) and analysed using the Swedish National Food

database (version 99.1). The milk products of interest were milk, soured milk (a traditional

milk product, “filmjölk” in Sweden), yoghurt, and cheese, in both reduced-fat and full-fat

varieties.

As regards dietary methodology it appears that two main concepts are often confounded or

misinterpreted concerning the diet of an individual and a collective. These are the concepts of

usual intakes and actual intakes. Observational nutritional epidemiology of large collectives is

26

24

primarily interested in an accurate estimation of actual intakes, eg 24-hour dietary recall.

Analytic nutritional epidemiology requires an accurate estimate of the past to present, ie usual

intakes. As regards the present work, based on genotyping for LP/LNP, which influences the

life-time exposure pattern, milk consumption, usual intakes are of less interest than actual

intakes. Genetic nutritional epidemiology using a functional genetic variant, already known to

stand proxy for lifetime exposure patterns (milk consumption, eg), do not need to readdress

the question of usual intakes of the food item in question for each and every individual. Intra-

individual variance, which is regarded both as random error or systematic error (because it

can differ between populations) as well as inter-individual variance is explained by the

genotype of the single subject or, the prevalence of the functional genetic variant in a

population. This is the reason why genetic nutritional epidemiology using a functional genetic

variant (eg Mendelian randomization) primarily needs an accurate estimate of the actual

intake of the food item in question.

3.2.2. Material and Methods ENCA

Sample size calculations resulted in a need of 1800 individuals, aged 6 to 75 years, necessary

for robust estimations of events rates of 10% to 20% and a precision ranging between 8% and

10% (α = 0.05) and to ensure a minimum of 100 individuals in every age group (10 years

intervals). The sample size was increased by 40% to ensure representativeness for the Canary

Islands. A stratified two-stage random sampling procedure was used weighted by the number

of inhabitants of each of the 32 sampled municipalities. Municipalities were the first unit

randomly sampled, and the last unit was the citizens registered in these municipalities. The

response rate was 69%. Non-participation (31%) included 17% voluntary non-participation,

14% involuntary non-participation (errors in census, changed domicile: 9%, absence or

impossibility to partake in the study: 6%).

To assess the nutritional status of the population two 24h-recall questionnaires were sent on

non-consecutive days prior to a visit at domicile and interview. Nutritional assessment also

included a food frequency questionnaire. We present in our sub-study data derived from the

24h-recall questionnaires. The “Tablas de Composición de Alimentos Españoles” (tables of

composition of Spanish food items) of Mataix et al. and the French tables of composition of

food items were used to transform data in energy intakes and single nutrients 79-80. ENCA also

27

25

included questionnaires about lifestyle and health status. Anthropometrics were determined

according to standardized procedures and blood pressure was taken. Sociodemographic and

lifestyle variables including age, sex, educational level, smoking, alcohol consumption and

physical activity were registered. Details on data collection have been published

elsewhere 88-89.

Seven hundred eighty two subjects participated in the biochemical analysis. The present study

is based on a sample of 551 adults aged 18 - 75 years (240 men and 311 women). Children

were excluded. Genetic analysis as regards the present study was performed at the University

hospital in Örebro, Sweden at the department of clinical chemistry.

ENCA revealed several particularities of the Canarian population of interest for our study.

The prevalence of obesity was in the Canary Islands 18 % higher than the national average

and one of the highest in Europe. Trans fatty acid consumption was on average higher than

estimates for the rest of Spain. With an intake of 6.8 g a day, trans fatty acid ingestion was at

levels comparable to the USA. The prevalence of diabetes type 2 is with 8%, 3% higher than

the national reference. Milk consumption in Spain is highest in the Canary Islands. The

average milk consumption is 322 ml a day and thus comparable or even higher than in some

countries in northern Europe. The same applies to the intakes potatoes and candy. Vegetable

intake in Canary Islands is the lowest of all Spanish autonomies.

3.2.3. Genomic Analysis

Genomic DNA was isolated from the EDTA whole blood samples from the individuals with

the QIAamp DNA Blood Mini Kit spin procedure. The DNA fragment spanning the -13910-

C/T polymorphic site was amplified using a biotinylated forward-primer

(5’ GGGCTGGCAATACAGATAAGATA-3’) and an unbiotinylated reverse-primer

(5’ AGCAGGGCTCAAAGAACAATCTA-3’). PCR amplifications were carried out in a

50 µL volume containing the forward and reverse primer, 1,25 units of Taq polymerase,

2.0 mmol/L MgCl2 and 0.2 mmol/L each of dGTP, dATP, dTTP, and dCTP. As templates

100 ng of the extracted genomic DNA was added. The PCR products were prepared for

Pyrosequencing according to a standard protocol (Pyrosequencing AB, Uppsala, Sweden)

using 25 µL amplicon captured by streptavidin-coated Sepharose beads and using the

28

26

Pyrosequencing Sample Preparation Kit (Pyrosequencing AB, Uppsala, Sweden); the applied

sequencing primer was: 5’-CTTTGAGGCCAGGG-3’. Sequencing was performed using a

PSQ96 SNP reagent Kit and a PSQ 96MA system (Pyrosequencing AB) PSQ96MA 2.0.1

software. This protocol is based on direct sequencing and yields unambiguous genotyping

results as well as sequence information beyond the SNP, which provides an internal control

for each sample 89. Simultaneous genotyping for the three polymorphic loci linked to LP

(LCT -13907 C>G, LCT-13910 C>T, LCT-13915 T>G) is routinely performed.

Pyrosequencing is thus a suitable method to detect LP/LNP not only in individuals of

European descent, but also of African or Middle East descent 30, 90.

4. Methodological Consideration

4.1. Hardy-Weinberg Equilibrium

Hardy-Weinberg equilibrium (HWE) describes a state in which genotype frequencies are

constant from generation to generation and in which genotype frequencies are product of

allele frequencies. This condition prevails in situations in which there are an absence of

mutations, migrations, non-random mating and environmental factors favoring particular

genotypes. In a paper published in 1908, Hardy wanted to disprove the Mendelian idea that

dominant genetic traits would have a propensity to appear in the whole population, while

recessive traits would tend to disappear. In the same year, Weinberg published a paper

proving the same idea. He later went a step further to be the first to apply it to human

populations. The original descriptions of HWE are an important landmark in the history of

population genetics 91 and it is now common practice to check whether observed genotypes

conform to Hardy-Weinberg expectations. Deviations from HWE can indicate inbreeding,

population stratification, and even problems of genotyping 92.

The prevalence of a genetic recessive or dominant trait can, when studied by genotyping

methods, be estimated directly as the prevalence of the homozygously mutated genotype. If

this type of trait is rare, the number of homozygotes in the studied sample will not be very

large, and the relative impact of sampling errors of various types can be expected to be rather

large when a prevalence estimate is based on the simple division term CC/(TT+CT+CC) as is

the case in the sub-study of this work. However, one can also arrive at an estimate of the

29

27

prevalence of the homozygous carriers of the mutated allele (q2) by utilizing all the

information that is contained in the Hardy-Weinberg Equilibrium, given the mathematical

relation p2 + 2pq + q2 = 1.00 between the prevalences of the mutated allele, eg q, and the

prevalence of the wildtype (unmutated) allele eg p. The advantage would be that the estimate

of q involves a much larger fraction of the population sample studied than the direct assay of

subjects in the sample that happen to have the genotype q2, since not only these homozygous

carriers of the mutated allele (q2) but also the heterozygotes (2pq) carry mutated alleles and

therefore the random error in the estimation of q2 from a HWE calculation will be relatively

smaller than that of the direct assay result by genotyping of the empirical prevalence of q2 in

the particular sample studied.

4.2. Mendelian Randomization

Since the carriage of the LCT -13910 C>T polymorphism is subject to the random assortment

of maternal and paternal alleles at the time of gamete formation (Mendel’s second law),

associations of LCT genotype with MS should not be subject to reverse causality, and largely

free from confounding by behavioral, physiological and socioeconomic factors related to both

exposure (milk consumption) and outcome (MS). This is the basic assumption of MR 61-62.

MR studies modifiable causes of disease in genetic epidemiology. A functional genetic

variant, in our study LCT–13910 C>T polymorphism, acts as proxy for modifiable lifetime

exposure patterns, i.e., milk consumption. The LCT–13910 C>T polymorphism is known to

influence the exposure pattern, milk consumption 20, 26, 64. According to Mendel’s second law

of independent assortment, the inheritance of one trait is independent of the inheritance of

other traits. This law suggests that inheritance is independent of –that is, randomized with

respect to –the inheritance of other traits. Thus, associations between genetic variants and

outcome are not generally confounded by behavioral, physiological or environmental

exposures. Hence, observational studies of genetic variants have similar properties to

intention to treat analyses in randomized controlled trials 61-63.

30

28

5. Main Results and Discussion

5.1. Study 1

The historical prevalence data for LNP in Sweden based on functional tests range between 1%

and at most 5% 64. Using genotyping for LCT –13910 C>T, we obtained an overall LNP

estimate of 14% among young Swedish citizens born in the 1980s, a markedly higher value

than the traditional figures ranging from 1 to 5%. In the elderly born in the 1920s we found a

prevalence of 6.8% for LNP. We proposed the following interpretation of these findings. The

frequency of the LCT –13910 C allele has increased from 2 sources: long-range immigration

from parts of the world with very high LNP prevalence, and a more long lasting immigration

of Caucasians from neighbouring European countries with a less dramatic but still markedly

higher LNP prevalence than the 6.8%. This higher LNP prevalence is seen in contrast to the

prevalence that seemed to have prevailed earlier in Scandinavian population samples as found

in studies from Norway, Finland, and Sweden (the elderly cohort in this study). This

significant shift in LNP prevalence in Sweden may have public health implications and should

therefore be monitored closely by health-planners and policy-makers 53. We used initially, and

in this sub-study, the term Adult type hypolactasia (AtH) to describe the status of LNP, which

we changed in the following sub-studies, because we considered LNP more appropriate.

31

29

5.2. Study II

The underlying hypothesis of this study was that LP and LNP might influence the

consumption of milk and milk products in children and adolescents, which could lead to

differences in body fat mass, between the different genotypes.

There was a statistically significant difference between overall milk and milk product

consumption in LP and LNP subjects. LNP was associated with a lower total consumption of

milk, soured milk and yoghurt in children and adolescents. Children and adolescents with the

TT and CT genotype consumed statistically significantly more reduced fat milk than LNP

subjects. Concerning the key question of this study, whether LP and LNP influence the

consumption of milk and milk products, which in turn could be associated with differences in

body composition between LP and LNP, no differences between LP and LNP of body

composition, especially in measures of body fat mass could be detected. In conclusion, LP

was linked to an overall higher milk and dairy intake, but was not linked to higher measures

of body fat mass in children and adolescents. Thus, an obesogenic effect of milk and milk

product intake could not be detected in the studied population 93. During the revision process

of this study a paper was published showing that a dietary pattern characterized by greater

milk intake was associated with increased BMI among preschool children 74. We took this

circumstance as an occasion to nurture the concept of adaptability in contrast to “one-size-fits-

all” public policies or solutions as regards public health nutrition 94. We hypothesize that

potential physiological effects of single food items, such as milk and milk products, may

differ also between countries due to their particular food and farming culture, agricultural

traditions and geographic as well as climatic singularities, among other potential factors.

The conception of this paper initially included the task to explore if an intermediate genotype

(LCT-13910 CT) were detectable. Since the finding of the trimodal distribution of lactase in

the small bowel traces back to the 1980s 13, we tested if the postulate of a mid-level-activity

of the enzyme would have an impact on milk and milk product intake using LCT genotypes as

proxy. We did not find mid-level intakes of milk and milk product in heterozygous

individuals.

32

30

5.3. Study III

Cow’s milk consumption is one of the most studied nutritional factors in relation to height

and growth rate in humans. Already in the early 1900s very well performed experimental

studies showed that cow’s milk supplementation in schoolchildren led to an increased

longitudinal growth 40-41. Today, a century later, the long-term effect of childhood milk

consumption on bone growth is still debated. There is, however, considerable evidence that

cow’s milk stimulates longitudinal growth 40-42, 46-47, 95 . Of particular interest are the ages

9 and 15 years, thus, before and after initiation of the pubertal growth spurt. Cow’s milk and

other dairy products are currently the primary source of calcium in Western diets and cow’s

milk avoidance has been linked to small stature 51-52. Swedish children and adolescents are of

specific interest for this study of potential links between milk consumption and body height

on account of the high prevalence of LP, and on account of the presence of one of the highest

consumption of cow’s milk and cow’s milk products in Europe and in the world. We found

that milk intake was a significant positive contributor to the observed variance in body height

when adjusted for parental height (β = 0.46; 95% CI: 0.040, 0.87 and p = 0.032). Also, the

LCT C>T-13910 polymorphism (LP vs. LNP), remained significant in the final model

(β = 2.05; 95% CI: 0.18, 3.92 and p = 0.032), showing a positive association of LP with

height. In addition, the statistical model we used was able to explain 90 % of the observed

variance in body height (adjusted R2 = 0.885). We hypothesize that non-caloric constituents in

milk such as IGF-I might influence the somatotropic axis in preadolescent and adolescent

children. Neither LP nor LNP was associated to higher energy intakes and higher milk

consumption did not translate in overall higher energy intakes. Figures 4 and 5 show the crude

(without the other regressors) linear relationship between milk intake and body height

stratified by Tanner score.

33

31

Figure 4: height (cm) by milk intake (g/d) in preadolescents (Tanner 1, 2), n=256

Figure 5: height (cm) by milk intake (g/d) in adolescents (Tanner 4, 5), n= 28

34

32

5.4. Study IV

In this study we examined an available population of the Canary Islands in Spain where, in

contrast to rest of Spain, there is a high prevalence of cardiovascular risk factors and one of

the highest cardiovascular mortality rates of the country. Furthermore, the highest milk

consumption in Spain is found in the Canary Islands. LP subjects consumed in average about

17% more milk compared to LNP subjects. LP subjects of the Canary Islands exhibited a 57%

higher risk to develop MS compared to LNP subjects. Interestingly, LP women showed a 93%

higher risk to develop MS compared to a 22% higher risk in LP men, after adjustment for the

main nutritional and environmental variables. We did not find, nevertheless, a significant

difference in milk consumption between women and men in their respective LP or LNP

group. This means that the quantity of milk consumed by women and men in their

corresponding LP or LNP group cannot explain the higher risk of LP women to develop MS

compared to LP men. We proposed the following rationale for this finding. Milk contains

hormonal constituents that influence endogenous hormones in sufficient quantities to have

biological effects in the consumer. One mechanism could be that milk consumption increases

insulin-like growth factor-I plasma levels in consumers 44, 47, 96-97. Furthermore, because of

contemporarily production methods, modern cow milk has a high content of sex steroids and

precursors of sex steroids such as progesterone (also bovine recombinant growth hormone is

used to increase milk production) 98. Progesterone and other steroid hormones have been

associated with markers of insulin resistance 99. Together these factors could shed light on the

observed higher risk of LP women to develop MS compared to LP men in the Canary Islands.

In addition, other sex steroids present in milk, especially testosterone, can influence plasma

levels of adiponectin plasma levels in women and men leading to changed insulin sensitivity.

35

33

6. General Discussion and Future Perspectives

Lactation has evolved gradually and a complex lactation system with elaborated milk

secretion was already in place 200 millions of years in the ancestors of mammals 100. This

said, it appears almost astonishing that LP emergence is dated only about 7,500 BC, thus

during Neolithic transition, which suggests gene-culture coevolution.

Since 2002 we are able to genotype for the SNP giving rise to LP. This circumstance led us to

investigate if the traditional prevalence figures for LNP in Sweden were up to date. We found

a 5.5 fold higher prevalence (14%) for LNP than current literature and noticed a secular trend,

which we interpreted as a result of the ongoing globalization leading to more heterogeneous

populations in industrialized countries. This also means that we can expect a 5.5 fold or even

higher prevalence of LNP individuals suffering symptoms of lactose intolerance, if milk and

some less processed milk products are consumed. Noteworthy is that LNP often continues to

be regarded by western scientific literature as a kind of disease that requires therapeutic

measures. This is not especially surprising given the fact that LP has been considered the

globally predominant condition and ‘normal’ status until the 1960s despite being 70% of the

world population LNP. The concept of a particular disease can, according to this, just

constitute a ‘social construct’ without anchoring in reality, evidence and last but not least

science. The term ‘Milk drinking syndrome’ has been coined to describe the phenomenon

when European experience is extrapolated to the other populations in the world 101.

LNP individuals are on average able to consume around 12g of lactose without symptoms of

lactose intolerance. This amounts to 250 ml fresh milk a day. However, consumed with meals

or intakes of lactose containing food items distributed through the day elevates the threshold

of tolerance to lactose. All together, this appears to be reasonable amounts of fresh milk

consumption given the circumstance that most processed milk products only contain a

fractional amount of lactose compared to milk, and can be consumed without symptoms of

lactose intolerance.

If disambiguation succeeds among professionals and policy makers a significant amount of

resources used to detect and treat LNP could be saved or utilized for diagnosis and treatments

of real-existing pathologies. Having said this, the existing diagnostic measures to detect

lactose intolerance in a broader sense are surely valuable and useful. A possible algorithm

36

34

about measures to be taken in cases of uncertainty or when differential diagnostics are

required has been described in the section: 1.1.2. Detection of LP and LNP.

Sweden belongs together with Finland to the countries with the highest per capita cow’s milk

intakes in the world. It was thus important for us to explore if milk consumption affects body

weight and body fat mass in children. Childhood milk consumption has become normative in

western countries surely influenced by two British scientific publications dated back to the

beginnings of the 20th century (1928 & 1929, respectively) 40-41. Weight gain in children was

regarded as beneficial at that time. In today’s completely different social settings, ‘high’ rates

of milk consumption in childhood continue to be associated to a healthy lifestyle in many

western countries. And, it is surely questionable if a ‘high’ consumption of whatever food

item can be considered as health promoting. However, it kept to be elucidated if milk

consumption affects measures of body weight, BMI and body fat mass in Swedish children;

thus, in a nutritionally replete country. The issue, whether milk consumption affects

negatively or positively children’s weight or body fat mass, constitutes an ongoing very

controversial topic. On account of these circumstances we performed sub-study 2. We did not

find any effect of milk intake on measures of body fat mass or body weight in preadolescent

and adolescent children in Sweden. Milk consumption showed neither an obesogenic effect

nor a weight reduction effect on the studied population. The same applies to the genotypes

LP/LNP, which also did not correlate with measures of body fat mass. However, the

drawbacks inherent to cross-sectional studies have to be kept in mind. We are not able to infer

causality, and we would not too, if we would have found a positive or negative association.

Interventional studies or studies with higher power will be necessary to confirm or rebut our

findings. Since health policy should and can aspire to the most equitable and efficient delivery

of preventive services, and following the time-honored principle of ”first do no harm”, we

cannot derive any public health recommendation from these findings.

Based on the studies of Leighton41 and Orr40 about a century ago, we decided to explore

whether milk consumption continues to exert an effect on body height in children under

today’s completely different socio-economic settings. We also become aware of Takahashi’s

article published 1984, “Secular Trend in Milk Consumption and Growth in Japan” - Japan, as

a country without appreciable cow’s milk consumption or dairy culture until the 1950s.

37

35

To our surprise we found a positive association between milk consumption and body height in

children. Sweden with its long dairy tradition and high milk consumption together with one of

the highest prevalence of LP could appear as the worst eligible scenario to detect an effect of

milk on body height. This was also the case until the low figures for R2 led us to reconsider

our statistical model. Including adjustment for parental height in our model finally delivered

not only an acceptable R2 value but also showed a positive correlation of both milk intake and

LP with body height. We also detected that nobody before us with exception of one study in

adolescents had corrected for parental height when studying the relation of body height to

milk consumption despite a large body of publications on this issue. In addition, even in many

twin studies on longitudinal growth, the circumstance that 80-90% of body height is heritable,

has been neglected. However, given the design of our study we are only able to generate

hypotheses primarily. We hypothesize that milk consumption together with LP status affects

positively body height in children. Given the paradigm “larger is healthier” which we today

experience our results might a priori been perceived as a ‘positive’ message. This is however

not the case. We conclude that our results should be interpreted with caution and should serve

policy makers, professionals in public health and public health nutrition to await a more

complete understanding of the complex effects of milk consumption on long-term health. This

applies particularly to developing countries with complementary feeding practices, which are

not based on dairy farming. Milk consumption in childhood might exert both negative and

positive effects, since greater stature has been associated to higher risk for some cancers 102 .

Using the tenets of Mendelian randomization we explored in an available population of the

Canary Islands in Spain if LP/LNP genotypes show an association with the metabolic

syndrome (MS) defined according to the criteria of the International Diabetes Federation

(IDF). The ENCA study revealed a prevalence of metabolic syndrome of about 24% in the

Canary Islands, which is highest in Spain and similar to the figures in the USA. LP subjects

from the Canary Islands displayed a 57% higher risk to develop metabolic syndrome than

LNP subjects. Stratified for sex, LP women showed a significantly higher susceptibility to

develop metabolic syndrome than men (93% vs 22%). This sex-specific difference might be

in part attributable to the criteria we used to define metabolic syndrome. The IDF criteria are

characterized by being abdominal obesity a necessary requirement to define metabolic

syndrome and the most prevalent metabolic syndrome criteria in the Canary Island among

women was abdominal obesity and low HDL-cholesterol. Given the panoply of different

definitions of MS, the election of one definition might alter sex-specific differences in one or

38

36

the other direction. The same applies to the overall prevalence of metabolic syndrome in a

defined population. Using the WHO criteria to detect the metabolic syndrome in the Canary

Island showed a prevalence of metabolic syndrome of 28% and using the ‘Third Report of the

National Cholesterol Education Program’ (NCEP) the figure was 23% 103. However, as a

construct that denotes risk factor clustering for cardio-vascular disease metabolic syndrome

has been a useful paradigm.

We also applied the ATP III definition to explore if the LP/LNP genotypes showed an

association with metabolic syndrome in the Canary Islands. We found a significant

association of LP with metabolic syndrome and the same prevalence figure, which confirms

our results using the IDF criteria. We conclude that the possibility of higher susceptibility of

LP subjects towards the development of metabolic syndrome exists among the adult

population of the Canary Islands. As LP individuals also showed higher intakes of milk we

believe that this higher susceptibility might be modulated by higher milk intakes. The

singularities of the diet of the Canary Islands has been described previously, and also other

milk products than just fresh milk are consumed in higher amounts in the Canary Islands

compared to the rest of Spain. The recommendation that can be derived out of this study has

been already implemented in the Canary Islands by ENCA, which is a shift towards a more

Mediterranean style diet.

In summary, it would be important to explore how LP evolved in other parts of the world,

probably via convergent evolution and varying demographic dynamics in Middle and South

America, Asia and Africa. This information would help international health and food aid

organizations to meet the region’s nutritional requirements in a more differentiated way. It

also is necessary to study in more detail and verticality the long-term effects of milk

consumption. This applies not only to industrialized countries but also to developing

countries. Studies with higher power than the power we were able to deliver or intervention

studies might be promising prospects.

39

37

Acknowledgments ett stort tack till: Professor Peter Engfeldt, my supervisor, for all the support. Professor Torbjörn Nilsson for all the support. Örebro läns landsting, Örebro Universitet and Forskningskommittén for all the support. Professor Michael Sjöström and co-author Emma Patterson for all the support and the opportunity to work with EYHS. Professor Lluís Serra Majem and co-author Eva Elisa Alvarez León for all the support and the opportunity to work with ENCA. Professor Curt Tysk for all the support. Professor Lewis Burgess for all the support. Susanne Collgård for the helpful hand. Lars Breimer for advice and encouragement. Lovisa Olsson for all the support. Anders Magnusson for the help with statistics. My family, friends and colleagues for all the support.

40

38

References

1. Matthews SB, Waud JP, Roberts AG, Campbell AK. Systemic lactose intolerance: a new perspective on an old problem. Postgrad Med J 2005;81(953):167-73.

2. Savaiano DA, Boushey CJ, McCabe GP. Lactose intolerance symptoms assessed by meta-analysis: a grain of truth that leads to exaggeration. J Nutr 2006;136(4):1107-13.

3. Burger J, Kirchner M, Bramanti B, Haak W, Thomas MG. Absence of the lactase-persistence-associated allele in early Neolithic Europeans. Proc Natl Acad Sci U S A 2007;104(10):3736-41.

4. Bersaglieri T, Sabeti PC, Patterson N, Vanderploeg T, Schaffner SF, Drake JA, et al. Genetic signatures of strong recent positive selection at the lactase gene. Am J Hum Genet 2004;74(6):1111-20.

5. Cavalli-Sforza LL. Analytic review: some current problems of human population genetics. Am J Hum Genet 1973;25(1):82-104.

6. Hollox EJ, Poulter M, Zvarik M, Ferak V, Krause A, Jenkins T, et al. Lactase haplotype diversity in the Old World. Am J Hum Genet 2001;68(1):160-72.

7. Mulcare CA, Weale ME, Jones AL, Connell B, Zeitlyn D, Tarekegn A, et al. The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene (LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans. Am J Hum Genet 2004;74(6):1102-10.

8. Myles S, Bouzekri N, Haverfield E, Cherkaoui M, Dugoujon JM, Ward R. Genetic evidence in support of a shared Eurasian-North African dairying origin. Hum Genet 2005;117(1):34-42.

9. Simoons FJ. Age of onset of lactose malabsorption. Pediatrics 1980;66(4):646-8. 10. Auricchio S, Rubino A, Landolt M, Semenza G, Prader A. ISOLATED INTESTINAL

LACTASE DEFICIENCY IN THE ADULT. Lancet 1963;2(7303):324-6. 11. Dahlquist A, Semenza G. Disaccharidases of small-intestinal mucosa. J Pediatr

Gastroenterol Nutr 1985;4(6):857-65. 12. Sahi T, Isokoski M, Jussila J, Launiala K, Pyorala K. Recessive inheritance of adult-

type lactose malabsorption. Lancet 1973;2(7833):823-6. 13. Ho MW, Povey S, Swallow D. Lactase polymorphism in adult British natives:

estimating allele frequencies by enzyme assays in autopsy samples. Am J Hum Genet 1982;34(4):650-7.

14. Enattah NS, Sahi T, Savilahti E, Terwilliger JD, Peltonen L, Jarvela I. Identification of a variant associated with adult-type hypolactasia. Nat Genet 2002;30(2):233-7.

15. Swallow DM. Genetics of lactase persistence and lactose intolerance. Annu Rev Genet 2003;37:197-219.

16. Sahi T, Launiala K, Laitinen H. Hypolactasia in a fixed cohort of young Finnish adults. A follow-up study. Scand J Gastroenterol 1983;18(7):865-70.

17. Buller HA, Grand RJ. Lactose intolerance. Annu Rev Med 1990;41:141-8. 18. Tishkoff SA, Reed FA, Ranciaro A, Voight BF, Babbitt CC, Silverman JS, et al.

Convergent adaptation of human lactase persistence in Africa and Europe. Nat Genet 2007;39(1):31-40.

19. Enattah NS, Jensen TG, Nielsen M, Lewinski R, Kuokkanen M, Rasinpera H, et al. Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture. Am J Hum Genet 2008;82(1):57-72.

41

39

20. Enattah NS, Trudeau A, Pimenoff V, Maiuri L, Auricchio S, Greco L, et al. Evidence of still-ongoing convergence evolution of the lactase persistence T-13910 alleles in humans. Am J Hum Genet 2007;81(3):615-25.

21. Ingram CJ, Elamin MF, Mulcare CA, Weale ME, Tarekegn A, Raga TO, et al. A novel polymorphism associated with lactose tolerance in Africa: multiple causes for lactase persistence? Hum Genet 2007;120(6):779-88.

22. Xu J, Turner A, Little J, Bleecker ER, Meyers DA. Positive results in association studies are associated with departure from Hardy-Weinberg equilibrium: hint for genotyping error? Hum Genet 2002;111(6):573-4.

23. Kuokkanen M, Enattah NS, Oksanen A, Savilahti E, Orpana A, Jarvela I. Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia. Gut 2003;52(5):647-52.

24. Olds LC, Sibley E. Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element. Hum Mol Genet 2003;12(18):2333-40.

25. Rasinpera H, Kuokkanen M, Kolho KL, Lindahl H, Enattah NS, Savilahti E, et al. Transcriptional downregulation of the lactase (LCT) gene during childhood. Gut 2005;54(11):1660-1.

26. Suarez FL, Savaiano D, Arbisi P, Levitt MD. Tolerance to the daily ingestion of two cups of milk by individuals claiming lactose intolerance. Am J Clin Nutr 1997;65(5):1502-6.

27. Vesa TH, Marteau P, Korpela R. Lactose intolerance. J Am Coll Nutr 2000;19(2 Suppl):165S-75S.

28. Jarvela IE. Molecular diagnosis of adult-type hypolactasia (lactase non-persistence). Scand J Clin Lab Invest 2005;65(7):535-9.

29. Lehtimaki T, Hemminki J, Rontu R, Mikkila V, Rasanen L, Laaksonen M, et al. The effects of adult-type hypolactasia on body height growth and dietary calcium intake from childhood into young adulthood: a 21-year follow-up study--the Cardiovascular Risk in Young Finns Study. Pediatrics 2006;118(4):1553-9.

30. Nilsson TK, Johansson CA. A novel method for diagnosis of adult hypolactasia by genotyping of the -13910 C/T polymorphism with Pyrosequencing technology. Scand J Gastroenterol 2004;39(3):287-90.

31. Swagerty DL, Jr., Walling AD, Klein RM. Lactose intolerance. Am Fam Physician 2002;65(9):1845-50.

32. Heyman MB. Lactose intolerance in infants, children, and adolescents. Pediatrics 2006;118(3):1279-86.

33. Waud JP, Matthews SB, Campbell AK. Measurement of breath hydrogen and methane, together with lactase genotype, defines the current best practice for investigation of lactose sensitivity. Ann Clin Biochem 2008;45(Pt 1):50-8.

34. Bamia C, Orfanos P, Ferrari P, Overvad K, Hundborg HH, Tjonneland A, et al. Dietary patterns among older Europeans: the EPIC-Elderly study. Br J Nutr 2005;94(1):100-13.

35. Hjartaker A, Lagiou A, Slimani N, Lund E, Chirlaque MD, Vasilopoulou E, et al. Consumption of dairy products in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort: data from 35 955 24-hour dietary recalls in 10 European countries. Public Health Nutr 2002;5(6B):1259-71.

36. Ingram CJ, Mulcare CA, Itan Y, Thomas MG, Swallow DM. Lactose digestion and the evolutionary genetics of lactase persistence. Hum Genet 2009;124(6):579-91.

42

40

37. Ong KK, Preece MA, Emmett PM, Ahmed ML, Dunger DB. Size at birth and early childhood growth in relation to maternal smoking, parity and infant breast-feeding: longitudinal birth cohort study and analysis. Pediatr Res 2002;52(6):863-7.

38. Visscher PM, Hill WG, Wray NR. Heritability in the genomics era--concepts and misconceptions. Nat Rev Genet 2008;9(4):255-66.

39. Silventoinen K. Determinants of variation in adult body height. J Biosoc Sci 2003;35(2):263-85.

40. Leighton G, Clark ML. MILK CONSUMPTION AND THE GROWTH OF SCHOOL CHILDREN: SECOND PRELIMINARY REPORT ON TESTS TO THE SCOTTISH BOARD OF HEALTH. Br Med J 1929;1(3548):23-5.

41. Orr J. MILK CONSUMPTION AND THE GROWTH OF SCHOOL-CHILDREN :Preliminary Report on Tests to the Scottish Board of Health. The Lancet 1928;211(5448):202-3.

42. Hoppe C, Molgaard C, Michaelsen KF. Cow's milk and linear growth in industrialized and developing countries. Annu Rev Nutr 2006;26:131-73.

43. Wiley AS. Does milk make children grow? Relationships between milk consumption and height in NHANES 1999-2002. Am J Hum Biol 2005;17(4):425-41.

44. Berkey CS, Colditz GA, Rockett HR, Frazier AL, Willett WC. Dairy consumption and female height growth: prospective cohort study. Cancer Epidemiol Biomarkers Prev 2009;18(6):1881-7.

45. Du XQ, Greenfield H, Fraser DR, Ge KY, Liu ZH, He W. Milk consumption and bone mineral content in Chinese adolescent girls. Bone 2002;30(3):521-8.

46. Hoppe C, Molgaard C, Juul A, Michaelsen KF. High intakes of skimmed milk, but not meat, increase serum IGF-I and IGFBP-3 in eight-year-old boys. Eur J Clin Nutr 2004;58(9):1211-6.

47. Hoppe C, Udam TR, Lauritzen L, Molgaard C, Juul A, Michaelsen KF. Animal protein intake, serum insulin-like growth factor I, and growth in healthy 2.5-y-old Danish children. Am J Clin Nutr 2004;80(2):447-52.

48. Takahashi E. Secular trend in milk consumption and growth in Japan. Hum Biol 1984;56(3):427-37.

49. Zhu K, Greenfield H, Du X, Zhang Q, Ma G, Hu X, et al. Effects of two years' milk supplementation on size-corrected bone mineral density of Chinese girls. Asia Pac J Clin Nutr 2008;17 Suppl 1:147-50.

50. Zhu K, Greenfield H, Zhang Q, Du X, Ma G, Foo LH, et al. Growth and bone mineral accretion during puberty in Chinese girls: a five-year longitudinal study. J Bone Miner Res 2008;23(2):167-72.

51. Black RE, Williams SM, Jones IE, Goulding A. Children who avoid drinking cow milk have low dietary calcium intakes and poor bone health. Am J Clin Nutr 2002;76(3):675-80.

52. Stallings VA, Oddleifson NW, Negrini BY, Zemel BS, Wellens R. Bone mineral content and dietary calcium intake in children prescribed a low-lactose diet. J Pediatr Gastroenterol Nutr 1994;18(4):440-5.

53. Almon R, Engfeldt P, Tysk C, Sjostrom M, Nilsson TK. Prevalence and trends in adult-type hypolactasia in different age cohorts in Central Sweden diagnosed by genotyping for the adult-type hypolactasia-linked LCT -13910C > T mutation. Scand J Gastroenterol 2007;42(2):165-70.

54. Buckler JM, Green M. The growth of twins between the ages of 2 and 9 years. Ann Hum Biol 2008;35(1):75-92.

55. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High

43

41

Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 2001;285(19):2486-97.

56. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med 1998;15(7):539-53.

57. Balkau B, Charles MA. Comment on the provisional report from the WHO consultation. European Group for the Study of Insulin Resistance (EGIR). Diabet Med 1999;16(5):442-3.

58. Kahn R, Buse J, Ferrannini E, Stern M. The metabolic syndrome: time for a critical appraisal: joint statement from the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care 2005;28(9):2289-304.

59. Alberti KG, Zimmet P, Shaw J. The metabolic syndrome--a new worldwide definition. Lancet 2005;366(9491):1059-62.

60. Lusis AJ, Attie AD, Reue K. Metabolic syndrome: from epidemiology to systems biology. Nat Rev Genet 2008;9(11):819-30.

61. Davey Smith G, Ebrahim S. 'Mendelian randomization': can genetic epidemiology contribute to understanding environmental determinants of disease? Int J Epidemiol 2003;32(1):1-22.

62. Davey Smith G, Ebrahim S. What can mendelian randomisation tell us about modifiable behavioural and environmental exposures? BMJ 2005;330(7499):1076-9.

63. Ebrahim S, Davey Smith G. Mendelian randomization: can genetic epidemiology help redress the failures of observational epidemiology? Hum Genet 2008;123(1):15-33.

64. Sahi T. Genetics and epidemiology of adult-type hypolactasia. Scand J Gastroenterol Suppl 1994;202:7-20.

65. Elwood PC, Pickering JE, Fehily AM. Milk and dairy consumption, diabetes and the metabolic syndrome: the Caerphilly prospective study. J Epidemiol Community Health 2007;61(8):695-8.

66. Pereira MA, Jacobs DR, Jr., Van Horn L, Slattery ML, Kartashov AI, Ludwig DS. Dairy consumption, obesity, and the insulin resistance syndrome in young adults: the CARDIA Study. JAMA 2002;287(16):2081-9.

67. Pfeuffer M, Schrezenmeir J. Milk and the metabolic syndrome. Obes Rev 2007;8(2):109-18.

68. Jauhiainen T, Korpela R. Milk peptides and blood pressure. J Nutr 2007;137(3 Suppl 2):825S-9S.

69. Snijder MB, van der Heijden AA, van Dam RM, Stehouwer CD, Hiddink GJ, Nijpels G, et al. Is higher dairy consumption associated with lower body weight and fewer metabolic disturbances? The Hoorn Study. Am J Clin Nutr 2007;85(4):989-95.

70. Haug A, Hostmark AT, Harstad OM. Bovine milk in human nutrition--a review. Lipids Health Dis 2007;6:25.

71. Zemel MB. Role of dietary calcium and dairy products in modulating adiposity. Lipids 2003;38(2):139-46.

72. Lawlor DA, Ebrahim S, Timpson N, Davey Smith G. Avoiding milk is associated with a reduced risk of insulin resistance and the metabolic syndrome: findings from the British Women's Heart and Health Study. Diabet Med 2005;22(6):808-11.

73. Berkey CS, Rockett HR, Willett WC, Colditz GA. Milk, dairy fat, dietary calcium, and weight gain: a longitudinal study of adolescents. Arch Pediatr Adolesc Med 2005;159(6):543-50.

74. Wiley AS. Dairy and milk consumption and child growth: Is BMI involved? An analysis of NHANES 1999-2004. Am J Hum Biol 2010;22(4):517-25.

44

42

75. Poortvliet E, Yngve A, Ekelund U, Hurtig-Wennlof A, Nilsson A, Hagstromer M, et al. The European Youth Heart Survey (EYHS): an international study that addresses the multi-dimensional issues of CVD risk factors. Forum Nutr 2003;56:254-6.

76. Riddoch CJ, Bo Andersen L, Wedderkopp N, Harro M, Klasson-Heggebo L, Sardinha LB, et al. Physical activity levels and patterns of 9- and 15-yr-old European children. Med Sci Sports Exerc 2004;36(1):86-92.

77. Wennlof AH. Cardiovascular Risk Factors in Children. Stockholm, Sweden: Karolinska Institutet; 2005.

78. Wennlof AH, Yngve A, Sjostrom M. Sampling procedure, participation rates and representativeness in the Swedish part of the European Youth Heart Study (EYHS). Public Health Nutr 2003;6(3):291-9.

79. Mataix J MM, Martinez de Vitoria E editor. Tabla de composición de alimentos españoles, 3.edición, Universidad de Granada. Granada 1998.

80. Favier JC I-RJ, Toque C, Feinberg M., editor. Repertoire General des Aliments. Table de composition. Paris. Technique & Documentation, INRA, CINQUAL-REGAL; 1995.

81. Henriquez Sanchez P, Diaz Romero C, Rodriguez Rodriguez E, Lopez Blanco F, Alvarez Leon E, Diaz Cremades J, et al. [Biochemical assessment of nutritional status in the Canary Islands population (1998)]. Arch Latinoam Nutr 2000;50(1 Suppl 1):43-54.

82. Serra Majem L, Armas Navarro A, Ribas Barba L. [Food consumption and food sources of energy and nutrients in Canary Islands (1997-98)]. Arch Latinoam Nutr 2000;50(1 Suppl 1):23-33.

83. Serra Majem L, Cabrera Leon A, Sierra Lopez A. [Conclusions of the Canary Islands Nutrition Survey (1997-98). Foundations for a nutrition policy in Canary Islands]. Arch Latinoam Nutr 2000;50(1 Suppl 1):62-70.

84. Serra Majem L, Ribas Barba L, Armas Navarro A, Alvarez Leon E, Sierra A. [Energy and nutrient intake and risk of inadequate intakes in Canary Islands (1997-98)]. Arch Latinoam Nutr 2000;50(1 Suppl 1):7-22.

85. Wennlof AH, Yngve A, Nilsson TK, Sjostrom M. Serum lipids, glucose and insulin levels in healthy schoolchildren aged 9 and 15 years from Central Sweden: reference values in relation to biological, social and lifestyle factors. Scand J Clin Lab Invest 2005;65(1):65-76.

86. Slaughter MH, Lohman TG, Boileau RA, Horswill CA, Stillman RJ, Van Loan MD, et al. Skinfold equations for estimation of body fatness in children and youth. Hum Biol 1988;60(5):709-23.

87. Gutin B, Litaker M, Islam S, Manos T, Smith C, Treiber F. Body-composition measurement in 9-11-y-old children by dual-energy X-ray absorptiometry, skinfold-thickness measurements, and bioimpedance analysis. Am J Clin Nutr 1996;63(3):287-92.

88. Álvarez León E, Henríquez P, Serra-Majem L. Mediterranean diet and metabolic syndrome: a cross-sectional study in the Canary Islands. Public Health Nutrition 2006;9(8A):1089-98.

89. Henriquez P, Doreste J, Deulofeu R, Fiuza MD, Serra-Majem L. Nutritional determinants of plasma total homocysteine distribution in the Canary Islands. Eur J Clin Nutr 2007;61(1):111-8.

90. Nilsson TK, Olsson LA. Simultaneous genotyping of the three lactose tolerance-linked polymorphisms LCT -13907C>G, LCT -13910C>T and LCT -13915T>G with Pyrosequencing technology. Clin Chem Lab Med 2008;46(1):80-4.

45

43

91. Crow JF. Eighty years ago: the beginnings of population genetics. Genetics 1988;119(3):473-6.

92. Wigginton JE, Cutler DJ, Abecasis GR. A note on exact tests of Hardy-Weinberg equilibrium. Am J Hum Genet 2005;76(5):887-93.

93. Almon R, Patterson E, Nilsson TK, Engfeldt P, Sjostrom M. Body fat and dairy product intake in lactase persistent and non-persistent children and adolescents. Food Nutr Res 2010;54.

94. Solomons NW. Developmental origins of health and disease: concepts, caveats, and consequences for public health nutrition. Nutr Rev 2009;67 Suppl 1:S12-6.

95. Wiley AS. Consumption of milk, but not other dairy products, is associated with height among US preschool children in NHANES 1999-2002. Ann Hum Biol 2009;36(2):125-38.

96. Adebamowo CA, Spiegelman D, Berkey CS, Danby FW, Rockett HH, Colditz GA, et al. Milk consumption and acne in teenaged boys. J Am Acad Dermatol 2008;58(5):787-93.

97. Giovannucci E, Pollak M, Liu Y, Platz EA, Majeed N, Rimm EB, et al. Nutritional predictors of insulin-like growth factor I and their relationships to cancer in men. Cancer Epidemiol Biomarkers Prev 2003;12(2):84-9.

98. Collier RJ MM, Mclaughlin CL, Johnson HD, Baile CA. Effects of recombinant somatotropin (rbST) and season on plasma and milk insulin-like growth factors (IGF-I) and II (IGF-II) in lactating dairy cows. Domest Anim Endocrinol 2008(35):16-23.

99. Brismar K, Nilsson SE. Interrelations and associations of serum levels of steroids and pituitary hormones with markers of insulin resistance, inflammatory activity, and renal function in men and women aged >70 years in an 8-year longitudinal study of opposite-sex twins. Gend Med 2009;6 Suppl 1:123-36.

100. Lefevre CM, Sharp JA, Nicholas KR. Evolution of lactation: ancient origin and extreme adaptations of the lactation system. Annu Rev Genomics Hum Genet 2010;11:219-38.

101. Jordania J. Who asked the first Question? The Origins of Human Choral Singing, Intelligence,Language and Speech 2006.

102. Gunnell D, Okasha M, Smith GD, Oliver SE, Sandhu J, Holly JM. Height, leg length, and cancer risk: a systematic review. Epidemiol Rev 2001;23(2):313-42.

103. Boronat M, Chirino R, Varillas VF, Saavedra P, Marrero D, Fabregas M, et al. Prevalence of the metabolic syndrome in the island of Gran Canaria: comparison of three major diagnostic proposals. Diabet Med 2005;22(12):1751-6.

Publications in the series

Örebro Studies in Medicine

1. Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term sequelae from closed head injury.

2. Jansson, Kjell (2004). Intraperitoneal Microdialysis.Technique and Results.

3. Windahl, Torgny (2004). Clinical aspects of laser treatment of lichen sclerosus and squamous cell carcinoma of the penis.

4. Carlsson, Per-Inge (2004). Hearing impairment and deafness.Genetic and environmental factors – interactions – consequences. A clinical audiological approach.

5. Wågsäter, Dick (2005). CXCL16 and CD137 in Atherosclerosis.

6. Jatta, Ken (2006). Inflammation in Atherosclerosis.

7. Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects onMalignant Diseases in Childhood.

8. Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods.

9. Norén, Torbjörn (2006). Clostridium difficile, epidemiology and antibiotic resistance.

10. Anderzén Carlsson, Agneta (2007). Children with Cancer – Focusing on their Fear and on how their Fear is Handled.

11. Ocaya, Pauline (2007). Retinoid metabolism and signalling invascular smooth muscle cells.

12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry in children.

13. Eliasson, Henrik (2008). Tularemia – epidemiological, clinical and diagnostic aspects.

14. Walldén, Jakob (2008). The influence of opioids on gastric function: experimental and clinical studies.

15. Andrén, Ove (2008). Natural history and prognostic factors inlocalized prostate cancer.

16. Svantesson, Mia (2008). Postpone death? Nurse-physicianperspectives and ethics rounds.

17. Björk, Tabita (2008). Measuring Eating Disorder Outcome – Definitions, dropouts and patients’ perspectives.

18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery.

19. Parihar, Vishal Singh (2008). Human Listeriosis – Sources and Routes.

20. Berglund, Carolina (2008). Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals.

21. Nilsagård, Ylva (2008). Walking ability, balance and accidental falls in persons with Multiple Sclerosis.

22. Johansson, Ann-Christin (2008). Psychosocial factors in patientswith lumbar disc herniation: Enhancing postoperative outcome by the identification of predictive factors and optimised physiotherapy.

23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco products.

24. Hahn-Strömberg, Victoria (2008). Cell adhesion proteins in different invasive patterns of colon carcinoma: A morphometric and molecular genetic study.

25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α and Methylenetetrahydrofolate Reductase Genes as Determinants of Plasma Homocysteine Concentrations.

26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence, treatment seeking behaviour, experiences and perceptions among persons with and without urinary leakage.

27. Elfström, Peter (2009). Associated disorders in celiac disease.

28. Skårberg, Kurt (2009). Anabolic-androgenic steroid users in treatment: Social background, drug use patterns and criminality.

29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment – Prevalence, Measurement and Long-term Outcome.

30. Vumma, Ravi (2009). Functional Characterization of Tyrosineand Tryptophan Transport in Fibroblasts from Healthy Controls, Patients with Schizophrenia and Bipolar Disorder.

31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis from a virulence and immunogenic perspective that includes variations in novel vaccine antigens.

32. Allvin, Renée (2009). Postoperative Recovery. Development of a Multi-Dimensional Questionnaire for Assessment of Recovery.

33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia with Special Reference to Folate and Fibrinolysis.

34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide study of assiciated morbidity and mortality.

35. Söderqvist, Fredrik (2009). Health symptoms and potential effects on the blood-brain and blood-cerebrospinal fluid barriers associated with use of wireless telephones.

36. Neander, Kerstin (2009). Indispensable Interaction. Parents’ perspectives on parent–child interaction interventions and beneficial meetings.

37. Ekwall, Eva (2009). Women’s Experiences of Gynecological Cancer and Interaction with the Health Care System through Different Phases of the Disease.

38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistancein Neisseria meningitidis – phenotypic and genotypic characteristics.

39. Hammer, Ann (2010). Forced use on arm function after stroke. Clinically rated and self-reported outcome and measurement during the sub-acute phase.

40. Westman, Anders (2010). Musculoskeletal pain in primary health care: A biopsychosocial perspective for assessment and treatment.

41. Gustafsson, Sanna Aila (2010). The importance of being thin – Perceived expectations from self and others and the effect on self-evaluation in girls with disordered eating.

42. Johansson, Bengt (2010). Long-term outcome research on PDR brachytherapy with focus on breast, base of tongue and lip cancer.

43. Tina, Elisabet (2010). Biological markers in breast cancer and acute leukaemia with focus on drug resistance.

44. Overmeer, Thomas (2010). Implementing psychosocial factors in physical therapy treatment for patients with musculoskeletal pain in primary care.

45. Prenkert, Malin (2010). On mechanisms of drug resistance in acute myloid leukemia.

46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal Sphincters in Obese Patients.

47. Josefson, Anna (2010). Nickel allergy and hand eczema – epidemiological aspects.

48. Almon, Ricardo (2010). Lactase Persistence and Lactase Non-Persistence. Prevalence, influence on body fat, body height, and relation to the metabolic syndrome.