University of Groningen
Brain-selective nutrients in pregnancy and lactation
Stoutjesdijk, Eline
DOI:
10.33612/diss.146373942
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Publication date: 2020
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Stoutjesdijk, E. (2020). Brain-selective nutrients in pregnancy and lactation. University of Groningen. https://doi.org/10.33612/diss.146373942
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1
Introduction:
Abbreviations
1,25(OH)2D, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D; 1C, one carbon;
AA, arachidonic acid; AI, adequate intake;
ALA, acid alpha-linolenic acid; ARA, antirachitic activity; As, arsenic;
AT, adipose tissue; Br, bromide; Ca, calcium;
CAD, coronary heart disease; Cd, cadmium;
Cu, copper;
CYP27B1, 25(OH)D-1-α-hydroxylase; DBP, vitamin D binding protein; DHA, docosahexaenoic acid; DMEQ-TAD, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl) ethyl]-1,2-4-triazoline-3,5-dione; DRIs, Dietary Reference Intakes;
DWGV, drinking water guideline values; EAR, estimated average requirement; EFA, essential fatty acids; EFAD, essential fatty acid deficiency; EPA, eicosapentaenoic acid; EQ, encephalization quotient; FAC, functional adequate concentration;
Fe, iron;
GEE, generalized estimation equation; GOED, Global Organization for EPA and DHA Omega3s;
GW, gestational weeks; Hb haemoglobin; K, potassium; I, iodine;
ICP-MS, inductively coupled plasma mass spectrometry;
IF, intrinsic factor;
IO-, hypoiodite;
IOM, Institute of Medicine; LA, linoleic acid;
LCP, long chain polyunsaturated fatty acids;
LC-MS/MS, liquid chromatography-tandem mass spectrometry; LOQ, limit of quantification; Ma, manganese;
Mg, magnesium;
MMA, methylmalonic acid; Mo, molybdenum; Na, sodium; nd, not detectable;
NIS, sodium/iodine symporter; NTD, neural tube defects; P, phosphorus;
PP, postpartum;
PPARs, peroxisome proliferator-activated receptors; PTAD, 4-phenyl-1,2,4-triazoline-3,5-dione; PTH, parathyroid hormone; PTHrP, parathyroid-hormone related peptide;
RAE, retinol activity equivalents; RBC, erythrocyte;
RCTs, randomized controlled trials; RDA, recommended dietary allowance; RE, retinol equivalents;
Se, selenium;
SOD, superoxide dismutase; TC-II, transcobalamin II;
TSH, thyroid-stimulating hormone; UL, tolerable upper intake level; US-EPA, United States Environmental Protection Agency;
WHO, World Health Organization; ya, years ago;
Zn, zinc
Brain-selective nutrients
One of the most remarkable features that distinguish us from other animals is the large size of our brain compared to total body size, also named the encephalization quotient (EQ). Compared with the cat (EQ=1.0)1, our brain/body ratio is about 7.6 times higher (EQ=7.4-7.8), followed by the dolphin (EQ=5.3) and monkeys and apes, notably our closest extant relative, the chimpanzee (EQ=2.5), with whom we share a common ancestor some 6 million years ago (ya). Our brain growth started about 2.5 million ya at the time of Homo
habilis and Homo erectus (Figure 1). A major question is ‘what made our brain grow’ from
the about 400 mL then to the current 1,300-1,500 mL now, and why didn’t it occur as well in our closest extant relative, the chimpanzee, with a current adult brain volume of about 400 mL? Such knowledge is important, since it might provide us with information on what particular foods, nutrients, and especially nutrient-combinations, are needed for optimal brain functioning, assuming that brain growth and functioning coevolved. Energy sources and essential nutrients are, of course, important for the functioning of each organ, but it has become clear that certain nutrients are particularly important for the proper functioning of our brain. This has led to the concept of ‘brain-selective nutrients’, first coined by Professors Stephen Cunnane, Michael Crawford and Leigh Broadhurst2-7. These nutrients comprise at least: iodine, selenium, iron, zinc, copper, vitamins A and D, vitamin B12 and the fish oil fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)4. According to Cunnane: ‘Brain selective nutrients are a collective term encompassing those nutrients needed especially for normal human brain development, and although they are important for brain, these nutrients aren’t specific to the brain; they are used elsewhere in the body as well. If the requirement for any one of the brain selective nutrients is not met at the correct stage of development, permanent retardation results’7. Each of these brain-selective nutrients is abundantly present in the so called ‘land-water ecosystem’, (see below) where our Palaeolithic ancestors are likely to have hunted and gathered from 2.5 million to 10,000 ya; the so called Palaeolithic Era. It is in this era that our genes adapted to the environment to ultimately provide us with a large brain according to the principles of Darwin’s ‘adaptation to the conditions of existence’, implying that knowledge on the ecosystem to which they were exposed in that era is key to the understanding of who we are and what our relatively large brain needs. The underlying mutations are of course important, but without the necessary nutrients, such mutations would eventually disappear by natural selection. Interestingly, a similar brain growth occurred in the above mentioned dolphin8, with whom we share a common ancestor some 95 million ya and who, after inhabiting the land, returned to the sea about 50 million ya. All of this occurred long before the brains of the ancestral dolphin and humans started to grow, the dolphin prior to the human lineage, suggesting that close contact with (sea) water is needed for such an event to occur8,9.
Figure 1. Human brain growth during evolution.
Obtaining a clear picture of our subsistence diet over the past 2.5 million years is of course an illusion, but one may try to reconstruct by combining information from many disciplines, including the Paleo-environment, comparative anatomy, biogeochemistry, archaeology, anthropology, (patho)physiology and epidemiology. It is commonly accepted that most of our evolution occurred in Africa10, notably in the East African Rift Valley. This Valley is a geographical trench, where two tectonical plates drift apart, stretching from Tanzania into Ethiopia and exhibiting a fair amount of volcanic activity that adds new minerals, such as iodine, to the local environment11,12. The East Rift is some 22-25 million years old and constituted an important habitat for Homo and its ancestral Ardipithecines and Austalophecines. South-Africa is, however, also a major archaeological excavation site13, but as a matter of fact our ancestors’ remains have been found all over Africa, such as those of the 316,000 years old anatomically-less-modern Homo sapiens found in a cave at Jebel Irhoud-Morocco14-16. As Hublin of the Max Planck Institute for Evolutionary Anthropology explained: ‘There is no Garden of Eden in Africa, or if there is, it is of the size of Africa’16. The currently oldest anatomically modern Homo sapiens, i.e. Homo sapiens idaltu, is about 160,000 years old and was found in Herto-Ethiopia near the middle part of the Awash river17. This Middle Awash has produced some 300 excavation localities and is the only place in the
world where fossils have been discovered spanning the entire hominin1 evolution. One of them, the 3.2 million years old ‘Lucy’, an Australopithecus from Haddar-Ethiopia, is among the widely known. Importantly, all of the remains in the Rift Valley were found in the vicinity of water18,19, whether fresh or salt (lake) water, in contrast to the savanna20. Remains of shallow-water animals, notably catfish and cichlids, turtles, frogs and crocodiles were found in the vicinity of the 5.8 million years old Ardipithecus Ramidus from the Middle Awash21, who is considered to have been an omnivore. Another important Rift-Valley excavation site at Lake Turkana-Kenya produced fossils of various mammals and bovidae, but also of bony fish, catfish, crocodile and turtle of which various carried cut-marks, attributed to the activity of hominins predating Homo erectus and taking place some 1.95 million ya22. Hunting and gathering activities by hominins on the savannah, a widespread hypothesis referring to the foraging activities of our ancestors, is rather unlikely. It is difficult to hunt animals in the wild savannah, even with modern weapons. An upright position while hunting in a flat open plain is not the favoured posture for success, and the daily need of at least 2 litres of fresh water, as required in the hot African savannah, would have been difficult to fulfil. The current view, based on genetics, is that Homo sapiens may derive from South Africa23, where excavations in e.g. the coastal caves at Pinnacle Point are still on going. These caves show e.g. evidence of shellfish consumption about 164,000 ya24. Shellfish (like oysters, Table 1) are rich sources of brain-selective nutrients, notably iodine, selenium, iron, zinc, copper, vitamin B12 and the fish oil fatty acids EPA and DHA. Easily harvested seaweed at low tide is a rich source of iodine and selenium (Table 1), while fish livers (Table 1) contain high amounts of vitamin A, vitamin D, and the other brain-selective nutrients. It has been estimated that eating 680, 800, 900, 500 and 300 g of shellfish, respectively, is needed to meet the daily requirements for iodine, iron, copper, zinc and selenium7. It is, however, unlikely that shellfish provided the only source of energy, e.g. because of its high protein content and the associated danger of protein poisoning29. Seaweed might have been a source of folate (Table 1), but is unlikely to have been the only dietary source. A diet from the land-water ecosystem might have lowered the requirements of both folate and choline, because of the local abundance of betaine, which is the oxidized (carboxylic) form of choline (an alcohol). Folate, choline and betaine find a common function in 1C metabolism in which a methyl group from 5-methyltetrahydrofolate (via vitamin B12-dependent methionine synthase and vitamin B2 -dependent methionine synthase reductase) or from betaine (via zinc--dependent betaine
1 Hominin – the group consisting of modern humans, extinct human species and all our immediate ancestors (including
members of the genera Homo, Australopithecus, Paranthropus and Ardipithecus); Hominid – the group consisting of all modern and extinct Great Apes (that is: modern humans, chimpanzees, gorillas and orang-utans plus all their immediate ancestors
homocysteine methyltransferase) is transferred to homocysteine for the regeneration of methionine25. Methionine regeneration from homocysteine with betaine is confined to the kidneys and the liver, and spares the catabolic use of choline as a precursor of betaine26,27. Choline is uniquely needed for the synthesis of acetylcholine (a neurotransmitter) and phosphatidylcholine (an abundant membrane phospholipid). In a similar manner the use of betaine as methyl donor also spares the expenditure of 5-methyltetrahydrofolate and the cofactors vitamins B2 and B12 that are needed in this reaction, and thereby saves the use of folate for its unique functioning (folate cycle) in the synthesis of purines and pyrimidines, both being building blocks for nucleic acid synthesis. Betaine is an osmolyte28, which is an intracellular compound that maintains osmotic pressure, notably upon abiotic (salt) stress. Not surprisingly, betaine is abundant in the land-water ecosystem, with high amounts occurring in e.g. shrimp (218-219 mg betaine/100 g;29,30, some seaweeds31 and plants growing close to the seashore to maintain osmotic balance, such as known of spinach (600-645 mg betaine/100 g;29,30). Mollusks and crustaceans living in shallow depths use betaine as one of the most abundant osmolytes and plants, such as the afore mentioned spinach32 and sugar beets, notably the wild ancestor of all beet crops33, accumulate organic osmolytes, such as betaine, when subjected to abiotic (salt) stress34,35. We hypothesize that betaine is, or has been, abundant at the South African coast and also in the vicinity of the salt lakes located in the Rift Valley, both considered to be the cradles of the current
homo sapiens.
It has been suggested that the exploitation of the local resources from the sea not only drastically changed social and technological behaviour to a society structure with e.g. small scale food production, that is unusual for hunter-gatherers36, but also saved Homo sapiens from extinction due to the harsh climatic conditions following its emergence in Africa37. This would imply a genuine ‘bottleneck’ in Homo’s recent evolution, suggesting that all current Homo sapiens are descendants from a population that lived at the South African Coast and survived the catastrophe of a long glacial stage known as Marine Isotope Stage 6 (MIS6) lasting from 195,000 to 123,000 ya37. It is also possible that human populations living at the South African coastal region were the only survivors of a decade or more of volcanic winter that followed the eruption of the Toba volcano in Sumatra-Indonesia about 74,000 ya38. Consumption of shellfish from sea or fresh water was not a trait unique for that period, but also occurred much earlier by Homo erectus, about 430,000-540,000 ya in Trinil Mid-Java, Indonesia39.
Ta bl e 1 . B ra in s el ec tiv e n ut rie nt s i n s ea fo od . N ut rie nt D im en sio n M ol lu sk s, o ys te r, ea st er , w ild , r aw Co nt en t (p er 1 00 g ) 40 Se aw eed , la ve r, r aw Co nt en t (p er 1 00 g ) 41 Sea w ee d: nor i, re d a lga e P. ten er a Co nt en t (p er 1 00 g d ry w ei gh t) 42 Se aw eed : Re d a lga e P. ha it an en sis Co nt en t (p er 1 00 g d ry w ei gh) 42 Co d, l iv er , ca nn ed Co nt en t (p er 1 00 g ) 43 H ad do ck , l iv er oil Cont en t (p er 1 00 g ) 44 Fi sh o il, c od live r Co nt en t (p er 1 00 g ) 45 RD A , A I ( IO M ; M , 31 -5 0 y ) 46 -5 0 Io din e μg 10 9 - 1 60 31 0, 80 0 ± 4 24 24 0, 70 0 ± 3 65 50 0 40 0 0 15 0 Se le niu m μg 19 .7 0.7 20 ,4 00 ± 10 3 12 ,6 00 ± 9 8 63 .5 0. 005 0 55 Iro n mg 4. 61 1. 8 18 ± 0. 20 3 70 .0 5 ± 0. 23 7 2.1 0. 074 0 8 Zin c mg 39. 3 1. 05 0. 06 0. 06 0 11 Copp er mg 2. 85 8 0. 26 4 0. 007 0. 007 0 0.9 Ch oli ne mg 65 10 .4 55 0 1 Fo la te μg 7 14 6 0 0 0 40 0 Vi ta m in A (R A E) μg 13 26 0 5,1 00 60 ,000 30 ,000 90 0 Vi tami n D μg 1 0 10 0 50 0 25 0 15 Vi tami n B 12 μg 8. 75 0 10 .6 0 0 2.4 EPA + D H A mg 313 80 9, 411 23 ,0 39 *Pe hr ss on e t a l. A m J Cli n N ut r 2 01 6 51; F oo d S ta nd ar d A us tr alia , N ew Z ea la nd 52 ,1; A I
1
Homo sapiens left Africa some 70,000-100,000 ya to become the only current Homo
species in the world. The local ‘conditions of existence’ caused humans to evolve into the present five races53, as adaptations to e.g. ultraviolet radiation54, but also many other environmental conditions, such as climate, altitude and notably infectious agents55,56. What factors exerted selection pressure57,58 is difficult to disentangle, since climate changes with latitude, but so does biodiversity, including pathogen diversity59. Infection by microorganisms is likely to have exerted very strong selection pressure, and still does, such as e.g., witnesses by the many mutations conferring some protection from malaria parasites60,61. Lactose persistence62, intuitively considered an advantage because it enables the intake of macro- and micronutrient rich milk, and amylase copy number variation63 for enhanced starch digestion starting high in the gastrointestinal tract64, might as well be adaptations to promote (rapid) simultaneous uptake of glucose, sodium and water to prevent life-threatening dehydration following vomiting and diarrhoea caused by exposure to mycotoxins on grains (introduced by the agricultural revolution), and exposure to zoonoses (that came along with animal domestication)65.
Whatever the genuine environmental factors, recent selection since the agricultural revolution introduced little genetic variation when compared to the already existing variation in the first Homo sapiens as a group66. For instance: genetically, two members of the same race are likely to differ more strongly from each other than the average member of their race differs from the average member of another race. In other words: genetically we are remarkably similar, which does not trivialise important genetic differences (such as polymorphisms, copy number variation) aiming at ‘adaptations to local environmental conditions’. The high incidence of skin cancer in white people who migrated to Australia is just one of the many examples reminding us that changes in environmental conditions to which humans became adapted may coincide with various health issues of the current time. The underlying ‘dysfunctional’ genes that received the blame are often referred to as ‘disease susceptibility’ genes67, which is a bizarre notion in the light of the evolutionary theory. It masks the influence of our current lifestyle in which >70% of colon cancer and stroke, >80% of coronary heart disease and >90% of type 2 diabetes mellitus can be prevented if we have more attention for specific aspects of diet, overweight, physical inactivity and smoking68. With a generation time of about 25 years, and only mild selection pressure that predominantly affects health at post-reproductive age, post agricultural revolution human populations are unlikely to have adapted their genes rapidly by natural selection following a sudden dietary change that does not cause acute deficiency or toxicity with severe short-term adverse reproductive consequences. The ensuing ‘mismatch’ between our Palaeolithic genes and this new, self-chosen, environment, and lifestyle in general, constitutes the basis of the so called ‘mismatch hypothesis’ that is fundamental to the discipline of ‘evolutionary medicine’69-75.
The Out-of-Africa diaspora is considered to have occurred via the coast76, with inland movements in e.g. the Americas occurring from there, notably by following rivers77. This suggests continuing availability of brain-selective nutrients from the land-water interface. Some evidence for the exploitation of food from the land-water ecosystem following ‘Out-of-Africa’ comes from shellfish fossils attributed to the activity of Neanderthals (who arrived earlier in Europe and are not in the Homo sapiens lineage) in the Bajondillo Cave (Malaga, Spain) dated 191,000-130,000 ya78, and the consumption of molluscs, seal, (scavenged) dolphin, and fish by Neanderthals about 42,000 ya in Gibraltar-caves. Many of the recovered animal remains were found to carry cut-marks, such as discovered on a bone fragment of a Mediterranean seal79. Stable isotope studies of bones recovered from early modern humans in China, near Beijing, and dated 40,000 ya, revealed consumption of dietary proteins that to a substantial portion derived from the freshwater ecosystem, probably freshwater fish80. The oldest fish hooks, made from shells were found together with remains of a variety of pelagic and other fish species, in Jerimalai Cave in East Timor Indonesia, dated 42,000 ya81. There is a 1-meter long life-size sculpture of a male salmon in the Abri du Poisson rock shelter (Franco-Cantabrian region), dated 23,000 ya, and located in the valley of the Gorge d’Enfer (valley of the Vezere River), close to Les Eyzies-de-Tayac in the Dordogne, France82. A painting of a giant black fish, thought to be a halibut, about 1.5 m in length, and dated about 18,000 BC, has been found in the ‘Fish Chamber’ of the La Pileta Cave (Cueva de la Pileta, Province of Malaga, Andalucia, Southern Spain)83. Chemical analysis of food residues (d13C values of 16:0 and 18:0 fatty acids in recovered lipids) adhering to pottery from Torihama and Taisho (Japan), dated 15,000 to 11,800 years BP (the incipient, so called, Jomon period), unequivocally revealed processing of freshwater and marine organisms. Most of the 101 charred deposits that were analysed from locations across the major islands of Japan were derived from aquatic food84.
With the event of the agricultural revolution and the concomitant domestication of (land) animals, starting some 10,000 ya in Mesopotamia, our ancestors started to replace their original hunter-gatherer diet for a mostly terrestrial diet composed of an eventually limited number of cultivated crops, such as cereal grains85, and (products of) domesticated ruminants like sheep, goats and cows86,87, and fowl like chickens, ducks and geese. The transformation from hunter-gatherer to (cattle) farmer changed our diet in a drastic manner, but as matter of fact our lifestyle in general. The fossil record indicates a sharp change in the 13C content of collagen isolated from human bones in coastal Britain at the onset of the Neolithic (4,000 ya), suggesting a rather sudden shift from a marine-based to terrestrial-based diet, concomitant with the estimated arrival of the agricultural revolution at those locations88,89. Using a combination of lipid biomarkers, stable carbon isotope signatures of individual fatty acids preserved in cooking vessels from the coastal Eastern North Atlantic (mainland Britain, Scottish isles and the isles of Man and Ireland), together
with archaeozoological and human skeletal collagen bulk stable carbon isotope proxies, it was shown that early farmers rejected marine resources coinciding with the adoption of intensive dairy farming and the overwhelming use of dairy products. After an initial sharp decline in the exploitation of marine resources from 4,600-4,300 BC (Late Mesolithic, retrieval of shell middens, fishing gear) to 3,800-2,200 BC (Neolithic, introduction of pottery, cattle, sheep) there was a subsequent gradual increase in the use of marine resources from 2,200-800 BC (Bronze age, intensified farming. limited fishing) to 800 BC-1,400 (Viking, deep sea fishing, intensification of trade). This increase, however, did not restore the use of marine resources to the level of the late Mesolithic90,91. In line with abandoning a diet from the land-water ecosystem there was an increase of many diseases of civilization, attributed at least in part to lower intakes of brain-selective nutrients3-5,8,18. It is not a coincidence that three brain-selective nutrients, i.e. iron, vitamin A and iodine currently belong to the class of micronutrients exhibiting the most widespread deficiencies in both developing and developed countries92-94.
Contemporary genetic adaptations, notably those of very recent origin, derive from relatively mild selection pressure. Unlike in the past, there have been no environmental threats that drove humans to near extinction and consequently no ‘bottlenecks’. Such adaptations will consequently meet difficulty to reach fixation in the context of an exploding world population with an estimated number of only 5 million people 10,000 ya, that was followed by an apparently linear growth that genuinely took off from 254 million in 1,000 AD to 6 billion in 2,000, and the reach of an anticipated number of 9.4 billion in 205095. This population growth is attributable to less infectious disease (in notably the GI- and respiratory- tracts) because of public health measures in e.g. hygiene and immunization, less violence because of changing culture, less famine thanks to the agricultural revolution, and also the institution of secondary prevention via health care96. Positive selection may on the other hand have speeded up in the past 40,000 years, because in a large population there are simply more new spontaneous mutations and because large population numbers exhibit less genetic drift (in which a new mutation might get lost by the higher chance that comes along with small numbers)97. Whatever our genetic adaptive ability, it is unlikely that the majority of us have evolved mechanisms to cope with the continuing and still rapidly changing, self-chosen, environment and modern lifestyle, rendering it at the same time conceivable that our current lifestyle reduces our number of years without chronic disease98,99, also referred to as our chance of ‘healthy aging’. This ongoing tragedy, with consequences for individuals and the costs of health care, happens in the context of the recent increase in life expectancy, and may together be viewed upon as a ‘double burden of disease’ distinct from its original definition by the WHO as the ‘coexistence of undernutrition along with overweight/obesity or diet-related non-communicable diseases’.
The loss of brain volume (175 mL for women and 158 mL for men; i.e. the size of about one tennis ball) during the Holocene (11,700-present)100 may relate to the reduced intake of brain-selective nutrients, and so may the seemingly rapid increase in psychiatric diseases, e.g. depression, notably in younger cohorts101 and young adolescents, although this epidemic is often attributed to the social media and insufficient sleep102. Also the apparent loss of 14 IQ points since the Victorian Era103 may be a consequence. Although not conclusive in this sense, it has been shown that ‘a poor diet’ is associated with a comparatively more rapid decline of brain (hippocampal) volume in 60-64 years old subjects104, while overweight and obesity are associated with more rapid loss of brain white-matter volume at later age105. One of the links between poor diet and a lifestyle with less brain-selective nutrients might derive from low long chain polyunsaturated fatty acid (LCP) intake, notably the fish oil fatty acids EPA and DHA. It is e.g. known that in adults the erythrocyte (RBC) omega-3 index (i.e. EPA+DHA) relates to total brain volume and hippocampal brain volume106, and that male babies born to mothers receiving a 600 mg DHA daily supplement from 23 gestational weeks (GW) until pregnancy end have larger head circumference, total brain, cortex, corpus callosum and whole grey matter107. It is clear that Homo sapiens is highly attracted to water, such as e.g. described by Elaine Morgan in her book ‘The Aquatic Ape Hypothesis: The Most Credible Theory of Human Evolution’ (1997)108. ‘The theory postulates that humans evolved through an aquatic stage, prior to Homo habilis and Homo erectus, during which time our ancestors lived largely in water’. The theory has both strengths and weaknesses109. Even today, over 50% of the world’s population lives closer than 3 km to a surface freshwater body, over 70% lives closer than 5 km to water and only 10% lives further than 10 km away110. Populations in Australia, Asia, and Europe live closest to water, 40% of Americans live close to the seashore111 while this latter applies to an even higher percentage of Australians112.
In the next paragraphs we will discuss the importance of some brain-selective nutrients to health and especially the development of a healthy brain.
The importance of nutrition prior to conception and during pregnancy and lactation.
The first 1,000 days, from conception to 2 years of age, are considered to constitute a critical window of growth and development. Maternal nutrition prior to conception and during pregnancy and lactation provides the embryo, fetus and breastfed infant with the essential nutrients for brain development, healthy growth and an optimal immune system113. Excessive or insufficient nutrient status in the period of conception, pregnancy and lactation may cause irreversible disturbances in the offspring’s brain development, or offspring physical growth, which leads to suboptimal cognitive performance in school,
but can also render them more susceptible to infection and disease. Figure 2 depicts the human development during the first 40 weeks of life and the important nutrients during the various stages.
Figure 2. The function and timing of micronutrients that affect outcome in offspring. Reprinted with
permission of Springer Nature: Nature Reviews Endocrinology, Micronutrients deficiencies in pregnancy worldwide: health effects and preventation. Gernand A.D., Schulze K.J., Stewart C.P., West K.P., Christian P. 2016114.
It has been shown that the affected offspring also has an increased risk of later conditions and diseases like obesity, type 2 diabetes, hypertension, coronary heart disease, chronic lung and kidney disease, musculoskeletal disorders, some cancers and mental illness115-117. As adverse exposures during early development shape the body´s responses to later
challenges, it is important to promote a healthy parental lifestyle, preferable during the entire life, but certainly starting before conception.
Folate deficiency is probably the best-known example to illustrate the potentially ill consequences of nutrient deficiencies on brain development. Folate is required for nucleotide and DNA synthesis to support cell division. It is also involved in 1C metabolism (see above). Intrauterine folate insufficiency strongly increases the risk of neural tube defects (NTDs)118,119. These are congenital malformations resulting from improper closure of the embryonic neural tube. The two most common NTDs are anencephaly and spina bifida. The central nervous system is formed early in embryonic life and folds itself to become a tube. Closure of the tube occurs in the fourth week of post-conception119. Therefore, an adequate maternal folate status in the first weeks of pregnancy is necessary to provide adequate infant levels and thereby prevent NTDs119,120. Food fortification with folic acid (the synthetic form), is estimated to prevent approximately 1,300 NTD-affected births annually in the United States121. Pre-conceptional supplementation with folic acid, alone, or as a part of a multi-vitamin, has been shown to reduce the risk of NTDs by 35-75%119,122. Other examples of lifestyle-interventions supporting healthy pregnancies are weight loss in obese women123, leading to increased natural conception rates124,125, and healthy eating habits, physical activity and self-monitoring of blood sugar levels by pregnant women with gestational diabetes to improve their own health and that of their babies, at least in part by the reduction of macrosomia126.
The placenta plays a key role in normal fetal growth and development. It provides the fetus with oxygen, nutrients and removes carbon dioxide and other waste products. The placenta also metabolizes certain nutrients in maternal or fetal blood and has an important function in fetal protection from injury, xenobiotics, infections and maternal diseases127,128. Micronutrient deficiencies may induce a pro-inflammatory state, and influence placental development and function, leading to impaired fetal growth129. Multi-nutrient supplements have been shown to reduce the incidence of low birth weight and small-for-gestational-age infants when supplied to women at high risk of deficiencies. In the first trimester of pregnancy the basic organ structures of the infant are established during embryogenesis, (2-8 GW). Important micronutrients for organogenesis are vitamins A and E, magnesium, iron, zinc and copper. The central nervous system and fetal brain are developed in the first post-conceptional weeks for which iron, zinc, copper, iodine, folic acid, choline, vitamin B12, vitamin A, vitamin D and DHA+EPA are important. Later in gestation, micronutrients are needed for organ size and function. In the second and third trimester the fetus grows and accumulates nutrient stores114.
Neonatal stores reflect both duration of gestation and maternal micronutrient status. The neonatal stores are crucial for ensuring optimal supply of micronutrients when breast milk, as a primary source of infant nutrition, does not contain adequate amounts of micronutrients. Low milk nutrient contents may occur in spite of an adequate maternal status. Building adequate infant stores seems especially important to prevent the use of certain nutrients by unfavourable bacteria in the gastrointestinal tract. Iron, for which mother-to-child transport seems safer during pregnancy than lactation, is a well-known example. Vitamin B12 might be another130,131. It is estimated that during pregnancy, 7 mg iron/day is transferred to the fetus132 to secure fetal iron stores, while breast milk only provides about 0.27 mg/day49, of which only 50% is bioavailable. Milk iron is tightly bound to milk proteins, notably lactoferrin, which contributes to the antimicrobial activity of human milk. The iron-binding proteins transferrin and lactoferrin restrict the amount of available ionic iron in body fluids to 10-18 M. For the binding of vitamin B
12 to intrinsic factor that is 10-15-10-9 M133. These concentrations are insufficient to support bacterial growth134. During the first 4 to 6 months after birth, iron becomes mobilised from the stores built during fetal life. Consequently the requirement of exogenous iron is virtually zero. After 6 months, the stores have been utilized and exogenous iron is needed to meet the infant requirement. In 7-12 months old infants, the daily iron need is estimated at 0.7 mg/day, of which 0.2 mg/day is required to replace losses. Low maternal status is likely to preclude the building of sufficient infant stores in utero114 and also preterm babies might have low stores. The World Health Organization (WHO) advices mothers to exclusively breastfeed their infants during the first six months of life135. Breast milk has advantages over formula milk. Not only does breastfeeding increase the bond between mothers and their infants, it also improves the infant’s immune system135. The composition of breast milk changes during the lactation period, to meet the infant’s changing needs. The physiological background encouraged the notion that breast milk is the “perfect food for infants”136, as popularized to “breast is best”. However, when comparing breast milk from all over the world, some similarities, but also many differences in composition are noted. Nutrients in breast milk derive from maternal stores, or may be synthesized by the lactocytes in the breast. Smaller amounts derive directly from the maternal diet137. Since nutrients in breast milk may derive from different sources, they can be classified into two groups.
Group I milk nutrients become rapidly and/or substantially reduced by maternal depletion. Maternal supplementation increases their breast milk concentrations and thereby improves infant status. Examples are: thiamin (vitamin B1), riboflavin (vitamin B2), vitamin B6, vitamin B12, choline, retinol, vitamin D, selenium, iodine and EPA and DHA. An adequate maternal diet during lactation is important to allow sufficient intakes of these nutrients by the infant137-139. Group II milk nutrients derive from their synthesis in the breast or from maternal
stores. In contrast to group I nutrients, those in group II, are considered relatively unaffected by short-term maternal intake or status. When maternal intake is below the amount secreted into milk, the mother becomes gradually depleted, and the infant turns into a parasite. Examples are folate, calcium, iron, copper and zinc140.
This classification of nutrients into groups I and group II may need reconsideration, as it could very well be possible that the breastfed infant depends on its own tissue stores (e.g. of vitamin D, iron and vitamin B12) to maintain adequate status. It might be hypothesized that the group I milk nutrients have been abundantly available during hominin evolution, precluding the need to tightly regulate their milk outputs. For instance by evolving transport systems with high concentrating ability as seen for e.g. iodine. In addition, abundances may have caused the possibility to create adequate stores during fetal life for e.g. vitamin B12. The close relation between maternal and infant nutritional status implies that the requirement (RDA. AI; see below) for infants could be best derived from (breast milk and blood) samples collected from life-long adequately fed mothers. However, since lifestyle has drastically changed during the past 200 years, it is nowadays difficult to define these requirements by studying ‘apparently healthy and well-fed’ mothers who are randomly selected from a population75.
To provide nutritional guidelines and to serve as a scientific basis for the development of food guidelines, the Institute of Medicine (IOM) developed Dietary Reference Intakes (DRIs). These DRIs are specified on the basis of age, gender and lifestyle and cover a wide range of nutrients141,142. The values and definitions that comprise the DRIs are:
• Estimated Average Requirement (EAR): “Reflects the estimated median requirement and
is particularly appropriate for applications related to planning and assessing intakes for groups of person”.
• Recommended Dietary Allowance (RDA): “Derives from the EAR and is intended to cover
the requirement for 97-98 per cent of the population”.
• Tolerable Upper Intake Level (UL): “Highest average intake that is likely to pose no risk”. • Adequate Intake (AI): “Used when an EAR/RDA cannot be developed. The AI is an average
intake level based on observed or experimental intakes”.
From the definition of the DRIs, it may be derived that EARs and RDAs are merely applicable to populations, and not individuals. Furthermore, these DRIs have been established for well-fed humans. Dietary needs of malnourished subjects or subjects with pre-existing micronutrient deficiencies are expected to be higher. During pregnancy, DRIs are determined by assessing the physiological requirements to support a healthy pregnancy. DRIs in pregnancy are, however, difficult to establish due to plasma volume expansion and
other pregnancy adaptations. Therefore, the recommendations are usually extrapolations from estimates from DRIs for adults, as adjusted for fetal nutrient accumulation and maternal demand to support tissue accretion and metabolism114. For lactating women, the DRIs are extrapolated from estimates from non-pregnant counterparts and adjusted for the average amounts secreted into breast milk142 For infants, the IOM established AI by using average intakes of full-term infants who are exclusively breastfed and are born to apparently healthy, well nourished mothers. It is unknown to what extent this approach results in the establishment of the actual requirements of nutrients in populations. In Table 2 we summarize the EARs, RDAs and AIs for micronutrients at different life stages and describe the methods that have been used to establish these for 0-6-month-old infants, and pregnant and lactating women. It is clear that for most nutrients, the DRIs are higher in pregnancy and lactation than for non-pregnant or lactating women of childbearing-age. In the next paragraphs we discuss the function, changes during pregnancy and intakes of vitamin B12, vitamin D, some essential fatty acids & long chain polyunsaturated fatty acids and some essential and toxic elements during pregnancy, lactation and early infant life.
Ta bl e 2 . D ie ta ry R efe re nc e I nt ak es o f t he I ns tit ut e o f M edi ci ne f or mi cr on ut rie nt s m ean t f or w om en an d t hei r 0 -6 -m on th -o ld i nf an ts , t og et he r w ith t hei r de riv at ion 46 -4 8, 50 ,14 1, 14 3-14 7 Li fe s ta ge Crit eria fo r s et tin g: Mic ro nu tr ie nt D im en si on Inf an ts : 0 -6 -mo nt h ol d Fem al es : 19 -5 0 y ea rs Pr egnan t w om en : 19 -5 0 y ea rs Lac ta ting w om en : 19 -5 0 y ea rs F em al es 1 9-50 y ear s: Pr egnan cy EAR RDA EAR RDA EAR RDA Vi tami n A 1 μg /d ay 40 0* , a 50 0 70 0 55 0 770 90 0 1, 30 0 c Co mp ut at io nal anal ys e t o a ss ur e ad eq ua te b od y s to re s o f v ita m in A A ss ur e a de qua te fe tal v itami n A s to re s Vi tami n B 12 μg /d ay 0. 4* , a 2. 0 2.4 2. 2 2. 6 2.4 2. 8 c M ai nt ai n ha em at ol ogi cal an d s er um vi tami n B 12 s ta tu s i n i nd iv id ua ls w ith p er ni ci ou s ana em ia in remi ss io n; endo ge no us B12 lo st i n b ile c or re ct in g f or bi oa va ila bil it y A bs or pt io n a nd u til iz at io n o f v ita m in B 12 , a ss ur e ad equa te fe tal v itami n B 12 s to re s, Vi tami n D μg /d ay 10 b 10 15 10 15 10 15 d Bo ne m ai nt enan ce N o e vi de nc e a n i nc re as ed i nt ak e is n ec es sa ry , b as ed o n o bs er va tio na l s tu di es w hi ch s ho w ed no e ffe ct o n m at er na l 2 5( O H )D l ev el s o n f et al ca lc iu m h om eo st as is o r s ke le to n o ut co m e Ome ga -3 -f at ty ac ids: E PA + D HA N ot e st ab lis he d; G O ED a dv ic es 7 00 m g EP A + D H A d ur in g p re gn an cy a nd l ac ta tio n Pot as siu m m g/d ay 40 0* , a 2, 60 0* 2,9 00 * 2, 80 0* , e Bal an ce s tu di es M edi an p ot as si um in tak es C al ci um m g/d ay 20 0* , a 80 0 1, 000 80 0 1, 000 80 0 1, 000 d Bal an ce s tu di es N o e vi de nc e a n i nc re as ed i nt ak e is n ec es sa ry So di um m g/d ay 11 0* , a 1, 50 0* 1, 50 0* 1, 50 0* Lo w es t s od iu m i nt ak es f ro m s od iu m t ria ls ; b al an ce s tu di es N o e vi de nc e a n i nc re as ed i nt ak e is n ec es sa ry Pho sphor us m g/d ay 10 0* , a 58 0 70 0 58 0 70 0 58 0 70 0 d Re la tio ns hi p b et w ee n s er um P i an d ab so rb ed in ta ke N o e vi de nc e a n i nc re as ed i nt ak e is n ec es sa ry M agn es ium m g/d ay 30 * , a 26 5 32 0 29 0-30 0 35 0-3 60 255 -26 5 31 0-32 0 d Bal an ce s tu di es Ex tr ap ol at io n o f a du lts (S er um M g / I nt ra ce llu la r M g / B al an ce s tu di es / m ag ne si um t ol er an ce t es t / Pr eg na nc y ou tc om e)
1
Ta bl e 2 . ( Con tin ue d) Li fe s ta ge Crit eria fo r s et tin g: Zi nc m g/d ay 2* , a 6. 8 8 9. 5 11 10 .9 12 c Fa ct or ia l a na ly sis o f z in c l os se s a nd re qu ire m en ts f or g ro w th , a s w el l a s fr ac tio nal a bs or pt io n A dd iti on al a ve ra ge d ai ly r at es o f z in c ac cum ul at io n b y m at er nal an d emb ry on ic /f et al tis su es i n t he 4 th quar te r o f p re gnan cy Iro n m g/d ay 0. 27 * , a 8.1 18 22 27 6.5 9 c D iv id in g t he r eq ui re d a m ou nt o f ab so rb ed i ro n b y t he f ra ct io na l ab so rp tio n o f d ie ta ry i ro n, e st im at ed t o b e 18 p er c en t f or a du lts f or t he t yp ic al N or th A m er ic an di et A dd iti on al i ro n d ep os ite d i n t he f et us a nd re la te d t is su e a nd i ro n u til iz ed i n e xp an si on o f ha em ogl ob in m ass Copp er μg /d ay 20 0* , a 70 0 90 0 80 0 1, 000 1, 000 1, 30 0 c C han ge s i n a c omb ina tio n o f b io ch emi cal in di ca to rs r es ul tin g f ro m v ar ie d l ev el s o f copp er in ta ke Es tim at io n o f a m ou nt s o f c op p er t ha t m us t b e ac cum ul at ed d ur in g p re gnan cy Io di ne μg /d ay 11 0* , a 95 15 0 16 0 220 20 9 29 0 c Io di ne a ccum ul at io n an d t ur no ve r Io di ne b al an ce , f et al th yr oi d r equi rem en t, sup p lem en ta tio n v er su s m at er nal s ta tu s Se len ium μg /d ay 15 * , a 45 55 49 60 59 70 c In ta ke n ee de d t o m ax im iz e t he a ct iv it y of p la sm a s el en op ro te in g lu ta th io ne p ero xi da se A ddi tio nal fe tal re qui rem en ts : t o s at ur at e f et al se le nopr ot ei ns M an gan es e m g/d ay 0. 003 * , a 1. 8* 2. 0* 2. 6* , e In su ffi ci en t a m ou nt o f d at a t o s et a n EA R; A I w as s et b as ed o n m ed ia n i nt ak es re p or te d f ro m t he U .S . F oo d a nd D ru g A dm in is tr at io n T ot al D ie t A ddi tio nal fe tal re qui rem en t + e xt rap ol at io n fro m a du lts M ol yb den um μg /d ay 2* , a 34 45 40 50 36 50 c Bal an ce s tu di es Ex tr ap ol at io n f ro m a du lts Th is t ab le p re se nt s R ec om m en de d D ie ta ry A llo w an ce s ( RD A s) i n o rd in ar y t yp e a nd A de qu at e I nt ak es ( A I) i n o rd in ar y t yp e f ol lo w ed b y a n a st er is k (*) . 1 A s r et in ol a ct iv it y e qu iv al en ts ( RA Es ): 1 RA E = 1 μ g r et in ol , 1 2 μ g β -c aro te ne , 2 4 μ g α -c aro te ne o r 2 4 μ g β -c ry pt ox an th in . T he R A E f or d ie ta ry p ro vi ta m in A c aro te no id s is t w o -f ol d g re at er t ha n r et in ol e qu iv al en ts ( RE ), w he re as t he R A E fo r p re fo rm ed v ita m in A is t he s am e a s R E. a m ea ns A I e st ab lis he d b y a ve ra ge b re as t m ilk c on ce nt ra tio ns ; b m ea ns A I is e st ab lis he d o n s up p le m en ta tio n n ee de d t o m ai nt ai n i nf an t 2 5( O H )D l ev el s ≥ 30 -5 0 n m ol /L ; c m ean s a ddi tio nal inf an t r equi rem en t; d m ea ns n o e vi de nc e w as f ou nd t ha t i nc re as ed i nt ak e a s n ec es sa ry ; e m edi an in tak e.
Vitamin B12
Vitamin B12 sources, metabolism, and function
Vitamin B12 is synthesized by certain bacteria and archaea, but not by plants and animals. From these micro-organisms vitamin B12 accumulates in animal tissues via the food chain. Good sources of vitamin B12 are meat, dairy and (shell) fish. In ruminants, vitamin B12 is synthesized in the stomach by vitamin B12-synthesizing bacteria. Vitamin B12 is absorbed in the intestine and stored in the liver, muscle or excreted into the milk. Pigs and chickens are omnivores, obtaining vitamin B12 from their diet. The vitamin B12 contents in products from ruminant animals are higher than those in products of omnivorous animals. In aquatic environments, bacteria live in symbiosis with phytoplankton, which become food for larval fish and bivalves. The bigger predatory fishes in the ocean food chain contain higher vitamin B12 levels. Most plants do not produce or require vitamin B12, although low levels of vitamin B12 have been found in some mushrooms and algae148.
Vitamin B12 is released from protein when entering the acid environment of the stomach and is subsequently bound by intrinsic factor (IF). The IF-vitamin B12 complex is absorbed in the small intestine by IF-receptor-(cubulin)-mediated endocytosis. Vitamin B12 is released from IF and is bound to transcobalamin II (TC-II). The transcobalamin II-vitamin B12 complex is transported in plasma and taken up in tissues by receptor-mediated endocytosis149. In the cells, vitamin B12 is an important co-factor in two enzymatic reactions: the one-carbon (1C) metabolic pathway and methylmalonic pathway, respectively (Figure 3). The 1C metabolic pathway is a series of biochemical reactions that are involved in amino acid and nucleotide metabolism. It is named 1C metabolism, because the reactions involve the addition, transfer, or removal of one-carbon groups. The 1C metabolism centres on the (re)methylation of homocysteine to methionine by methionine synthase, in which 5-methyltetrahydrofolate is converted to tetrahydrofolate, which is important for RNA- and DNA synthesis. The other enzymatic reaction takes place in the mitochondrion, where vitamin B12 is involved in the conversion of methylmalonyl-CoA to succinyl-CoA, by the enzyme methylmalonyl-CoA-mutase, which is important for fatty acid, cholesterol and lipid metabolism, energy metabolism and the synthesis of haemoglobin150. In brain, vitamin B
12 is important for myelin formation151.
Figure 3: One carbon metabolic pathways, vegetarian diets and effects of B12 insufficiency. X block; ll
‘secondarily’ inhibited; à stimulated; -| inhibited by metabolite. BHMT, Betaine-homocysteine S-meth-yltransferase; CPT1, Carnitine palmitoS-meth-yltransferase; CBS, Cystathionine-β-synthase; DNMT, DNA methyl-transferase; GNMT, Glycine N-methylmethyl-transferase; MCM, methylmalonyl-CoA mutase; MMA-CoA, Methyl-malonyl-CoA; MTR, Methionine synthase; MTHFR, methylentetrahydrofolate reductase; MS, Methionine Syntase; R Methyl acceptors, including adenosine and cytosine; R-CH3 Methylated acceptor; SAH, S-ad-enosyl homocysteine; SAM, S-adS-ad-enosyl methionine; THF, Tetrahydrofolate. Reprinted with permission from Springer Nature: European Journal of Clinical Nutrition, Vitamin B12: one carbon metabolism, fetal growth and programming for chronic disease. Rush E.C., Katre, P. Yajnik, C.S., 2014150.
Vitamin B12 during pregnancy and lactation
Maternal cobalamin deficiency is related to an increased risk of early and recurrent miscarriage, preterm birth, and low birth weight and may contribute to anaemia152. As vitamin B12 is important for the rapidly developing fetal nervous system, low maternal vitamin B12 status is, like folate, associated with NTD risk.
The course of vitamin B12 status during pregnancy is somewhat difficult to interpret. Widely employed parameters of vitamin B12 status are plasma/serum vitamin B12 concentrations, vitamin-B12 dependent concentrations of methylmalonic acid (MMA) and homocysteine and holotranscobalamin. In a controlled feeding study, assessing the effect of reproductive state on biomarkers of vitamin B12, it was found that serum vitamin B12 declines during pregnancy, reaching about 21% lower values in the 3rd trimester of pregnancy as compared to the non-pregnant state153. During pregnancy it has been found that holotranscobalamin concentrations fall between preconception and the first trimester and then remain relatively stable152, or remain similar to non-pregnant controls153, while MMA increases and homocysteine decreases153. These changes in vitamin B
12 biomarkers seem to be based on physiological changes rather than vitamin B12 deficiency. A combination of
hemodilution, hormonal changes and vitamin B12 transfer from mother to infant may decrease plasma vitamin B12 concentrations. Vitamin B12 recovers to normal values within 8 weeks postpartum154.
The infant’s postnatal vitamin B12 sources are its own liver stores and breast milk or formula. Infants born to vitamin B12 adequate mothers have liver vitamin B12 stores of ~25-30 μg versus 2-5 μg in mothers with inadequate vitamin B12 status155. Infant vitamin B
12 deficiency is a worldwide problem156. It may cause megaloblastic anaemia, microcephaly and neurological symptoms such as irritability, failure to thrive, apathy, anorexia, hypotonia and developmental delay157-159.
Occurrence of (sub)clinical vitamin B12 deficiency in pregnant women and infants
Maternal vitamin B12 insufficiency may be widespread since 7% of women of reproductive age exhibit low plasma vitamin B12, while 5% may be vitamin B12 deficient in early pregnancy and 10% thereafter160. At delivery, low vitamin B
12 status has been noted in 40% of UK Caucasian mothers and 29% of their offspring161, while 13% of Belgian mothers and 0% of their infants162 exhibited low vitamin B
12, respectively. Vitamin B12 deficiency is more prevalent in breastfed infants as compared to formula fed infants163,164.
RDA/AI of pregnant and lactating women and infants
Pregnant and lactating women have higher vitamin B12 needs than non-pregnant counterparts due to fetal and infant requirements. The IOM has set the RDAs at 2.6, 2.8 and 2.4 μg/day, respectively48. The DRIs for women of childbearing age are based on normal serum vitamin B12 (defined as ≥150 pmol/L), the estimated extra loss of 0.4 nmol/L vitamin B12 in subjects with pernicious anaemia in remission and an average fractional absorption of vitamin B12 of about 50%48. For pregnant- and lactating DRIs, the additional requirements for the fetus and infant are added. However, these DRIs might not be sufficient to maintain adequate vitamin B12 status153. Based on vitamin B
12-related biomarker studies, the nutrition societies of Germany, Austria and Switzerland recently raised their reference values for vitamin B12 intakes to 4.0, 4.5 and 5.5 μg/day for non-pregnant, pregnant and lactating women, respectively165.
For 0-6-month-old infants the IOM-AI is 0.4 μg/day. This AI is based on 9 milk samples of healthy 2 months postpartum lactating women in Brazil166 with a mean milk vitamin B
12 concentration of 310 pmol/L. Using an average intake of 780 mL/day167, this translates to the consumption of 0.33 μg/day, which was rounded to 0.4 μg/day by the IOM. The 0.4 μg/ day corresponds with a milk vitamin B12 concentration of about 378 pmol/L. The inverse relationship between urinary MMA and milk vitamin B12 at milk vitamin B12 concentrations
less than 362 pmol/L confirmed the adequacy of this recommendation164. However, the above-mentioned studies are based on small numbers of observations and the milk vitamin B12 concentrations have been measured with methods that may have incorrectly co-measured vitamin B12 analogues168,169. As a consequence, there is no well-established AI.
Aim of the vitamin B12 studies in this thesis
Currently there is a need for a well-established vitamin B12 AI, as derived from exclusively breastfed infants. In Chapter 2 we describe the outcomes of vitamin B12 measurements in mature milk of mothers living in various countries. The results were evaluated for ‘adequacy’ with the use of the 378 pmol/L vitamin B12/L estimate of the IOM, and also by employing a Functional Adequate Concentration (FAC), calculated as the geometrical mean of milk of mothers with ‘functional vitamin B12 adequacy’. The latter was derived from Vietnamese mothers exhibiting a plasma vitamin B12 >221 pmol/L and a plasma MMA <210 nmol/L, which, in combination, are considered to reflect an adequate vitamin B12 status170-172.
Vitamin D
Vitamin D sources, metabolism and function
Cutaneous synthesis of parent vitamin D3 by exposure to UVB is widely regarded as our principal source of vitamin D3. Vitamin D3 can also be obtained from animal foods, especially fish, whereas vitamin D2 can be found in fungi and plants, but may also be present in fish. However, the often-reported relatively low intake of vitamin D from fish is somewhat artificial, because Western cultures do not exhibit the habit to consume the vitamin D-rich fish liver (see Table 1). For instance, tuna liver may contain 32,500 µg vitamin D/kg and the fatty meat 37 µg/kg, while mackerel liver may contain 2,400 µg/ kg and the fillet 155 µg/kg173. In view of these figures, it may be questioned whether our (black) ancestors living in the land-water ecosystem found their principal vitamin D source in cutaneous synthesis. This may be important since the pharmacokinetics, body distribution, and therefore targets, of cutaneously-synthesized vitamin D are different from dietary vitamin D174,175. In addition, no attention has until now been paid to the ingestion of the 25-hydroxy metabolite of vitamin D [25(OH)D] from muscle meat, which functions as a 25(OH)D store176,177. This pool may have been an important, and at least five times more efficient177, dietary vitamin D source of hunter-gatherers in the past.
Following exposure of skin to UV-B from sunlight, pre-vitamin D3 is converted from 7-dehydrocholesterol to become thermally isomerized to vitamin D3. Once formed, vitamin D3 diffuses into the capillary beds to become transported by circulating vitamin D binding protein (DBP) and to a lesser degree albumin. In contrast to cutaneously synthesized vitamin D3, dietary vitamin D3 and vitamin D2 [together referred to as ‘parent vitamin D’], are
transported from the gastrointestinal tract to the liver by chylomicrons and subsequently by other lipoproteins174,178.
In the liver, vitamin D is converted to 25(OH)D by 25-hydroxylase (CYP2R1), although other organs can also perform this reaction. Compared with 25(OH)D, vitamin D is easily taken up by various organs and excreted into e.g. breast milk, due its lower affinity to DBP. 25(OH)D has a higher binding affinity for DBP, which may explain its longer half-life of 3 weeks, compared with the 24 hours of the parent vitamin D. Uptake of the DBP-25(OH)D complex is facilitated by megalin-cubulin-mediated endocytosis178. In the kidneys, but also in other organs like the placenta and the brain, 25(OH)D may become converted to active hormone 1,25-dihydroxyvitamin D [1,25(OH)2D] by 25(OH)D-1-α-hydroxylase (CYP27B1), as shown in Figure 4.
Figure 4. the metabolic processes providing vitamin D and its metabolites to various tissues in the body.
Tissue distribution of vitamin D and 25(OH)D based on simple diffusion (red arrows) and endocytosis (green arrows). Endocytosis requires the tissue-specific megalin-cubilin system, whereas simple diffu-sion is primarly controlled by the dissociation constant of the vitamin D compound for the VDBP. Bolder red lines indicate greater diffusion rates due to higher dissociation constant, t½, half-life. Reprinted with permission from Springer Nature: Bone Research, New insights into the vitamin D requirements during pregnancy. Hollis, B.W. Wagner, C.L. 2017179 This image is licensed under the Creative Commons
Attribu-tion 4.0, no changes were made. To view a copy of the Creative Commons license, please visit http:// creativecommons.org/licenses/by/4.0/.
To preserve adequate vitamin D status after the migration from Africa to higher latitudes, some adaptations took place. Skin depigmentation is likely an adaptation that enables vitamin D synthesis at low UVB exposure180. In addition, mobilization of parent vitamin D from adipose tissue181,182 and mobilization of 25(OH)D from muscle tissue176,183, might have enabled populations at higher latitudes to preserve an adequate status during winters, characterized by low UVB exposure and limited food sources.
The classical function of the vitamin D hormone lies in bone and calcium homeostasis, that is: bone health and augmentation of calcium absorption. 1,25(OH)2D is, however, also involved in the regulation of e.g. cell growth184,185. In addition, vitamin D is involved in e.g. inflammation, mitochondrial protein expression186 and has an unproven anti-oxidant effect187. It has been estimated that about 10% of the human genome is regulated directly and/or indirectly by the vitamin D endocrine system188, involving more than 160 pathways some of them linked to cancer, autoimmune disorders and cardiovascular disease189. Developmental vitamin D deficiency has been shown to impact the expression of 36 proteins in the adult rat brain with functions in neurotransmission and synaptic plasticity in neurons and astrocytes, and with oxidative phosphorylation, redox balance, calcium/ATP homeostasis and organelle transport in mitochondria190. Vitamin D signalling may influence brain development via its pro-differentiation and anti-apoptotic properties191 and may also play a role in fetal programming192. Many areas of the brain, including the amygdala, hippocampus, thalamus, cortex, and substantia nigra, express both vitamin D receptor and 1α-hydroxylase. Neurons and microglia may use the synthesized active hormone to regulate cell proliferation, differentiation, and survival193. Recently, developmental vitamin D deficiency was shown to impact recognition memory in rats and that this effect is independent of the applied time window of vitamin D deficiency194.
It was for long believed that 1,25(OH)2D was the only active metabolite, while parent vitamin D and 25(OH)D were merely considered to be pro-hormones. Recently, Gibson et
al.195 showed that vitamin D and its metabolites are effective stabilizers of the endothelium and can control ‘endothelial leak’. On an equal molar basis, parent vitamin D is more potent than 25(OH)D and 1,25(OH)2D in this respect179,195.
Vitamin D in pregnancy and lactation
Low vitamin D status in pregnant women is associated with increased risk of uteroplacental dysfunction196, bacterial vaginosis197, pre-eclampsia196, gestational diabetes197,198, intrauterine growth restriction199, preterm delivery197,200, spontaneous pregnancy loss201 and higher mother-to-child HIV transmission202. Vitamin D insufficiency during pregnancy is associated with lower offspring birth weight and postnatal growth197,199, dental cavities203, lower gross-motor development and fine-motor development at 30 months and lower social
development at 42 months, but not at older ages204, greater adiposity at 4 and 6 years205, mildly- or moderately- severe language impairment at 5 and 10 years206, reduced lung function and asthma at 6 years207, and lower peak bone mass at 20 years208.
Since the half-life of 25(OH)D is highest, plasma 25(OH)D is generally accepted as the best vitamin D status parameter. Widely employed cut-off values are <25 nmol/L for vitamin D deficiency, 25-50 nmol/L for vitamin D insufficiency and >50 nmol/L for vitamin D sufficiency46. However, many vitamin D experts consider 25(OH)D levels between 50 and 75-80 nmol/L as hypovitaminosis D, and between 75-80 and 250 nmol/L as vitamin D sufficiency209-211. During pregnancy, significant changes occur in vitamin D metabolism. From 12 GW, maternal serum levels of 1,25(OH)2D are two- to threefold higher compared to the non-pregnant state212. Also DBP increases during pregnancy as a response to the increasing oestrogen concentrations213,214. In a cross-sectional study with unsupplemented traditionally-living Tanzanian women with lifetime abundant sunlight exposure, we found higher 25(OH)D in pregnancy, but similar concentrations at 3 days and 3 months PP, when compared to non-pregnant counterparts215.
Postnatal vitamin D sources include stores, exposure to sunlight and breast milk or formula. Breast milk contains both the parent vitamin D and 25(OH)D, which are usually summed to the so-called antirachitic activity (ARA, in IU/L). Most breastfed infants in Western societies receive supplemental vitamin D to reach an adequate vitamin D status. Infant vitamin D deficiency is associated with low bone mass at 9 years216, autoimmune diseases including diabetes mellitus type 1 and multiple sclerosis217, acute lower respiratory tract infections218,219, autism and schizophrenia191 and asthma220,221.
Occurrence of (sub)clinical vitamin D deficiency in pregnancy and lactation, and infants
Vitamin D deficiency and insufficiency are worldwide problems222. Among the vulnerable groups are elderly people, persons with darker skin, subjects who cover their body and often also avoid direct sunlight, pregnant and lactating women and their exclusively breastfed infants222,223. Depending on definitions, it is estimated that the worldwide prevalence of vitamin D deficiency and insufficiency during pregnancy ranges from 8 to 100%224. For instance, the Dutch ‘Generation R’ study reported vitamin D deficiency (25(OH)D< 25 nmol/L) in 26%, and vitamin D insufficiency (25OHD ≥ 25 and <49.9 nmol/L) in 27%, at 20 GW. Cord blood samples showed 46% deficiency and 34% insufficiency225. Alarmingly, 50% of the non-European mothers in this study exhibited vitamin D deficiency at midgestation, while 75% of their offspring was deficient at birth225.
RDA/AI pregnant and lactating women and infants
The RDA for pregnant and lactating women is identical to that of non-pregnant adults up to 70 years. They range from 10 μg/day (Health Council of the Netherlands226) to 15 μg/ day (IOM46). However, the median dietary vitamin D intake of 19-30 years Dutch women is only 2.6 μg/day227. The Health Council of the Netherlands advices pregnant women to take a 10 μg/day vitamin D supplement, starting preferably before conception30. The current RDA of 10-15 μg/day might be insufficient to reach 50 nmol/L 25(OH)D in 97.5% of the women at the pregnancy end. This can be concluded from two recent randomized controlled trials (RCTs) conducted in pregnant women with median 25(OH)D levels of 55 nmol/L and 64-68 nmol/L at enrolment in the 2nd trimester in, respectively, New Zealand228 and Canada229. Still 7-12% of these women had 25(OH)D levels <50 nmol/L at 36 GW, after supplementation with 25-50 μg vitamin D/day.
The IOM AI for vitamin D for 0-6-month-old infants amounts to 10 μg/day46. This AI is based on some studies in Western countries showing that 10 μg/day maintains infant serum 25(OH)D at 40 to 50 nmol/L in the first postnatal year and thereby supports normal bone accretion46. A 10 μg/day intake translates to a milk ARA of 513 IU/L at an average mature milk consumption of 780 mL/day167. Breast milk ARA from Western mothers ranges from 8 to 331 IU/L230-232. It is unclear what vitamin D dose is needed to reach the IOM AI of 513 IU/L in milk. The only successful study in reaching the IOM AI was published by Wagner et al.233. They showed that supplementation of lactating women with 160 μg/day for 6 months increased milk ARA to 873 IU/L at the study end. However, although perfectly safe, with no adverse effects noted nor expected, a daily 160 μg/day vitamin D dose is well above the current upper limit of 100 μg/day234. Finally, a strategy merely aimed at the postnatal period provides no benefits for the mother and her developing child during pregnancy.
Aim of the vitamin D studies in this thesis
From an evolutionary point of view, it is puzzling why breast milk contains only low amounts of vitamin D. We therefore investigated milk ARA from mothers with lifetime abundant sunlight exposure. In Chapter 3.1 we investigated whether the milk ARA from mothers with different cultural backgrounds, living at different latitudes, reaches the AI. In Chapter 3.2 we investigated what dosages of vitamin D are needed to reach vitamin D adequacy during pregnancy and lactation by supplementing pregnant Dutch women with vitamin D (10, 35, 60 and 85 μg/day) from 20 GW up to 4 weeks postpartum (PP). We furthermore investigated whether the corresponding milk ARA reaches the AI.
Since data on parent vitamin D during pregnancy and lactation are lacking, we developed methods to measure parent vitamin D in plasma and adipose tissue, to gain a better
understanding of vitamin D metabolism during pregnancy and lactation. In Chapter 3.3 we describe the parent vitamin D concentrations in plasma and adipose tissue (AT) of pregnant and non-pregnant women and infants in traditionally living people in Tanzania, in relation to earlier published 25(OH)D data.
Essential fatty acids (EFA) and long chain polyunsaturated
fatty acids (LCP)
EFA sources, metabolism and functions
The omega-3 fatty acid alpha-linolenic acid (ALA) and omega-6 fatty acid linoleic acid (LA) are essential fatty acids (EFA), meaning that they should derive from the diet. They cannot be synthesized by the human body, and their deficiency causes disease. ALA and LA can be converted to LCP with 20 and 22 carbon atoms, by alternating desaturating and elongating enzymes that show preference in the order ω3FA> ω6FA> ω9FA235. The functionally of the most important metabolites of these LCP derive from DHA (22:6ω3), EPA (20:5ω3) and arachidonic acid (AA; 20:4ω6). Humans have limited capacity to synthesize EPA, DHA and AA236, even during pregnancy and lactation. A strong argument is the, for long known, postnatal decrease of infant LCP status, notably of DHA, following feeding with formula that do not contain LCP. Thus, EPA, DHA and AA are considered ‘conditionally EFA’, but some argue that these LCP should be regarded as the genuine EFA237. The exception is LA that has a function of its own in the limitation of transepidermal water loss238.
Vegetable oils, nuts and seeds are rich sources of ALA and LA. Meat, eggs and poultry are important sources of AA239, while fatty fish contain high levels of EPA and DHA. Lean and tropical fishes are usually low in EPA240, but this often quoted misconception may, like vitamin D, refer to the current habit to merely consume the muscle meat (see Table 1). Fish muscle only contains high percentages EPA and DHA in ‘fatty fish’ that in contrast to ‘lean fish’ store triglycerides between cells, as opposed to the liver. The food chain of EPA and DHA in fish starts with phytoplankton, where EPA can be found in the diatoms, and DHA in the (dino)flagellates241.
Sixty percent of the brain is composed of fat, in which LCP, notably DHA and AA, are abundant and play important roles in fetal and infant neurodevelopment242. In the retina, 50% of the fatty acids in photoreceptor membranes consist of DHA, stressing the importance of DHA for the human sight243. LCP are incorporated into membrane phospholipids and are important for fluidity, flexibility, permeability and modulation of membrane-bound proteins (lipid rafts)244,245. Furthermore DHA, EPA and AA, and their many prostaglandin-like highly potent metabolites are involved in a variety of processes, via their role in cell signalling and gene-expression by interaction with receptors in cell membranes
