METABOLISM OF VITAMIN D IN MAN AND IN ANIMALS

WITH SPECIAL REFERENCE TO SUN EXPOSURE AND RENAL FAILURE

by

Jan Wilske

METABOLISM OF VITAMIN D IN MAN AND IN ANIMALS WITH SPECIAL REFERENCE TO SUN EXPOSURE AND RENAL FAILURE Jan Wilske, Department of Nephrology, Sahlgrenska University Hospital, University of Gteborg, S-413 45 Gteborg, Sweden Abstract Vitamin D3 is formed in man and animals by radiation of the basal layer in the epidermis with sun light in the ultraviolet light range with wavelengths between 290 and 315 nm (UV-B). In the liver, Vitamin D3 is converted to calcifediol (25-OH-D3). It is known from investigations in Europe, Japan, USA and Antarctica that the amount of this vitamin D metabolite in the population varies with the season as a consequence of the seasonal variations in UV-radiation. Calcifediol is in turn hydroxylated to calcitriol (1,25-(OH)2-D3). This is the hormonal form of vitamin D3. Normally, the synthesis in the kidneys is the only one of systemic importance. Serum phosphate has a strong regulatory influence on the synthesis of calcitriol. In a Swedish normal population, there is a strong correlation between calcifediol serum levels and the sunny season, independent of other factors. In Antarctica during the summer, the plasma levels of calcifediol increased in both penguins (Pygoscelis papua) and humans in spite of the high latitude. Female southern elephant seals (Mirounga leonina) in Antarctica also increased in their plasma levels of calcifediol significantly when they spent their time on land for breeding or moulting. Sun light is the most important determinant of the serum concentrations of calcifediol. It shows a seasonal variation both in Sweden and in Antarctica and may be used as an index of UV-radiation. In spite of abundant sunlight signs of rickets are relatively common (39.3%) in 05 years-old-children from a low-income, suburban population in Uruguay. The children with rickets had lower serum concentrations of calcifediol, but still within the normal range. The intake of milk (and calcium) is lower in children 4 and 5 years old with signs of rickets. Regarding calcitriol patients with end-stage renal disease still have a production of this metabolite that can be stimulated by lowering serum phosphate. In patients treated with hemodialysis, calcium carbonate is an alternative to aluminium hydroxide as a binder of the alimentary phosphate. The synthesis of calcitriol in these patients could be increased by reduction of serum phosphate using calcium

carbonate but not aluminium hydroxide. A protein-reduced diet in patients with renal failure also lowers the serum phosphate and increases the serum levels of calcitriol after 3 months of treatment but not after 6 months. Key words: Antarctica, calcifediol, calcitriol, calcium carbonate, hemodialysis, penguins, protein-reduced diet, rickets, serum phosphate, southern elephant seals, Swedish normal population, ultraviolet light Gteborg, Sweden, 1995 ISBN 91-628-1499-0

This thesis is based on the following papers:

(I). Wilske J: Can plasma concentrations of 25-hydroxyvitamin-D3 (calcifediol) be used as an indicator of ultraviolet radiation and ozone layer thickness? Arctic Med Res 52:162-169 (1993).

(II). Wilske J, Arnbom T: Seasonal variation in vitamin D metabolites in southern elephant seal (Mirounga leonina) females at South Georgia. Submitted to Comp Biochem Physiol.

(III). Landin-Wilhelmsen K, Wilhelmsen L, Wilske J, Lappas G, Rosn T, Lindstedt G, Lundberg P-A, Bengtsson B-: Sunlight increases serum 25(OH) vitamin D concentration whereas 1,25 (OH)2D3 is unaffected. Results from a general population study in Gothenburg, Sweden (The WHO MONICA Project). Submitted to Eur J Clin Nutr.

(IV). Wilske J: Rickets in Uruguay, a field survey and biochemical findings. Submitted to Bull Panam Health Org.

(V). Wilske J, Bjrk S, Delin K: Calcium carbonate as a phosphate-binder in hemo-dialysis patients. Effect on the release of PTH and calcitriol. Scand J Urology Nephrol 26:51-54 (1992)

(VI). Wilske J, Attman P-O: Increase of calcitriol during treatment with proteinreduced diet in patients with renal failure. Nephron 66:421-425 (1994).

The Guanche (Wunchee) symbol is from the original residents of the Canary Islands. The symbol of light was very powerful and sacred to them. It was believed that this symbol was a protection against radiation.

CONTENTS Vitamin D in general...................................................................................... 9 Formation in the skin.................................................................................... 10 Ultraviolet light and the earth's ozone layer................................................. 12 Regulation of vitamin D3 metabolism..........................................................13 Calcifediol.....................................................................................................13 Calcitriol - The hormonal form of vitamin D3............................................. 15 Renal metabolism of vitamin D3.................................................................. 16 Calcium.........................................................................................................17 Parathyroid hormone.................................................................................... 17 Aluminium.................................................................................................... 17 The aim of the study..................................................................................... 19 Materials and Methods................................................................................. 20 Vitamin D in the Antarctic region................................................................ 20 Humans......................................................................................................... 20 Animals.........................................................................................................20 Clinical studies..............................................................................................23 A Swedish population...................................................................................23 Children in Uruguay..................................................................................... 23 Renal patients................................................................................................24 Analytical methods....................................................................................... 26 Statistical methods........................................................................................ 27 Results...........................................................................................................28 Correlation between vitamin D3 metabolites and sunlight at different latitudes...28 In Antarctica................................................................................................. 28 In a general Swedish population...................................................................33

Is there a connection between the calcium content in the diet and vitamin D3 metabolites and clinical signs of rickets?..................................................... 36 Control of serum phosphate by phosphate binders and the production of calcitriol ...................................................................................................................... 42 Discussion.....................................................................................................48 Sunlight and calcifediol................................................................................ 48 In penguins in Antarctica..............................................................................48 In humans in Antarctica................................................................................48 In elephant seals in Antarctica...................................................................... 49 A general population in Sweden...................................................................51 Rickets, calcium and the metabolism of calcifediol..................................... 52 Control of calcitriol synthesis in renal failure.............................................. 55 Notes on future research............................................................................... 59 Summary.......................................................................................................60 Conclusions...................................................................................................62 Acknowledgment.......................................................................................... 63

VITAMIN D IN GENERAL

The ancient Greeks are said to have made the observation that the people living in the northern countries had weaker skulls, that were more easily fractured by hand weapons, than the people that lived in the southern countries. In that case it is the first observation of the importance of sunlight for the strength of the skeleton. Homer and Hippocrates are also said to have described of rickets, but this is unproved (1). Soranus from Ephesus did the first published description of this disease when he worked as a medical practician in Rome during the time of Trajanus and Hadrianus (2). The next description was made more than 1500 years later at the beginning of the industrial revolution separately by Francis Glisson and Daniel Whistler. The latter published 1645 in Leyden a thesis with the title: "De morbo puerili Anglorum" from which the name "The English disease" is derived.

Already 1822 is was proposed that rickets was caused by lack of sun light (3). Later during the same century Palm showed that rickets was a small problem in children in China, India and Japan even if they were poor and had a bad nutrition. This was in contrast to the children of the middle class in central London and Manchester. Palm also proposed sun bath as treatment and prevention of rickets (4).

During the present century the cause of rickets was finally found. Mellanby could 1918 and 1919 describe a fat soluble substance in cod liver oil. Deficiency of this substance would produce rickets (5,6). Mellanby himself held that this substance

was vitamin A. Later, in 1922, it was clearly demonstrated that this factor was new substance that was named "vitamin D" (7).

However, ultraviolet light by itself without cod liver oil could also cure rickets (8,9). Also, ultraviolet irradiation on the skin and food stuffs converted an inert substance to vitamin D (10,11). So, vitamin D got its name because it was discovered as a nutritional factor that cured rickets. The name "vitamin" is in fact wrong because it is produced in the body by irradiation of the skin by sun light.

Formation in the skin

Vitamin D3 is formed in man and animals by radiation of the basal layer in the epidermis or the body surface with sunlight in the ultraviolet range with wavelengths between 290 and 315 nm. The synthesis starts by photolysis of 7dehydrocholesterol (provitamin D3) to a secosteroid (previtamin D3) (12). This is a thermally unstable substance that, under the influence of the body heat, isomerize in its double bonds to vitamin D3. At 37 C, 50% of the formed previtamin D3 has been converted to vitamin D3 after 1 day and after 3 days most of it is converted.

This process is temperature labile. Lowering of the temperature by 12 C reduces the formation rate by 50% and at freezing point there is practically no conversion. However, this endothermic process takes place in the epidermis near the dermocapillar vascular bed. Because of this there is very little change in the formation of vitamin D3 if the skin is exposed to strong cold or heat (13).

10

After single exposure of the whole body surface to a dose of ultraviolet radiation that produces a minimal sunburn (i.e. 1 minimal erythema dose = 1 MED), the concentration of vitamin D3 in the blood rises within the first 24-48 hours and is back to pre-exposure level after 7 days (14). An increase of the radiation dose to 4 MED gives proportionally higher peak concentrations of vitamin D3. On the basis of these and other experiments, the half-life in the skin has been calculated to be 48 hours and its production capacity to be 30 g/m2 at 1 MED (15).The melanin in the skin absorbs the sun's wavelength spectra between 290 and 700 nm (16) and functions as a sunscreen. Because of this it has been proposed that human tribes that live near the equator are dark pigmented to prevent too high a vitamin D production and that people have turned lighter when they have migrated north and south, from the equator, to be able to maintain a normal production of vitamin D (17).

In experiments of the same type as mentioned above, black and white volunteers have been UV-radiated with a dose equal to 1.5 MED in whites. In the white subjects the vitamin D3 serum concentrations rose to 30 times the base line level while the same dose in the black subjects did not give any change in the concentrations of this substance. If the radiation dose was increased 6 times for the black subjects (i.e. to 9 MED, which would give second-degree burns in whites) the serum concentrations of vitamin D3 also rose 30 times (18). The conclusion is that human beings of all races have the same capacity to produce vitamin D3 in their skin, but, as consequence of the filtering effect of the skin pigmentation, higher pigmentation necessitates higher intensity of UV-radiation to produce the same amount of vitamin D3. If a subjective measure of UV-exposure is used (MED), it is found that the same degree of erythema gives the same production of vitamin D (19,20).

11

Beside pigmentation, variations in the sunlight's energy content control the production of vitamin D3 in the skin (21). This means that the vitamin D-synthesis varies with the time of the day, season, latitude, height over sea level and ozone layer thickness.

Ultraviolet light and the earth's ozone layer

The influence of the seasonal stratospheric ozone depletion on the ultraviolet climate has been recognized during the last decade (22). The ultraviolet range of the sun's rays can be divided into 3 different bands, named UV-A, UV-B, and UV-C. This subdivision is somewhat arbitrary and varies depending on the scientific discipline. In photobiology these limits apply: UV-A 400-320 nm, UVB 320-290 nm, UV-C 290-200 nm. The limit 290 nm is important, because the earth's atmosphere stops rays of shorter wave-lengths reaching the surface of the planet (23). These shorter wavelengths are stopped by the earth's ozone layer, which is formed in the stratosphere at 25 to 100 km above the surface of the earth. Ozone is formed by short wave ultraviolet radiation below 240 nm. Further absorption of UV-light shorter than 320 nm transforms the ozone back to oxygen (24). As long as 20 years ago, scientists warned that chlorofluorocarbons (CFCS) could interfere with the normal balance of creation and destruction of ozone and lead to a thinning of the stratospheric ozone layer (25). Spring time ozone depletion over Antarctica has been observed for approximately 10 years. The first time this was observed in reality was in 1985 when a 50% depletion was recorded (26). A downward trend by 2-3% per year during the last 30 years has also been recorded over the northern hemisphere (27). UV-measurements are important to assess a possible influence of a changed UV climate in the biosphere. The first measurements with the double monochromatic spectrophotometer were done a

12

few years ago. A biological system, directly weighting radiation by its biological efficiency, seems to be a practical alternative.

Regulation of vitamin D3 metabolism

Vitamin D3 diffuses from the basal epidermis into the bloodstream, where it is bound to a specific carrier protein (vitamin D binding protein or Group-specific component) (28). This protein is a glucoprotein of the 1-globulin group with a molecular weight of approximately 68000 dalton and a serum concentration of 400 mg/ml in man.

Calcifediol

In the liver, vitamin D3 is converted to 25-hydroxyvitamin-D3 (calcifediol, 25-OH-D3) (29). The conversion is dependent upon cytochrome P-450 (30). Vitamin D3 itself lowers the conversion by 50% in vivo and in vitro (31,32). Calcifediol is the vitamin D3 metabolite with the highest serum concentration (1560 ng/ml) and it has a half-life of 3 weeks. Its concentration gives a good picture of the total pool of vitamin D in the body and indicates both surplus and lack.

As mentioned above, after radiation of white-skinned persons with 1 MED the serum concentration of vitamin D3 rose from 5 to 60 ng/ml within 24 hours and calcifediol rose by 30-50% within a week (14).

It is not clear whether calcifediol by itself has any systemic effect. In rickets and osteomalacia one generally finds, low serum concentrations of this metabolite

13

together with normal values for calcitriol. Other investigations support the opinion that 25-hydroxyvitamin-D3 has an intrinsic effect on calcium metabolism (33) and on renal control of phosphate (34).

Patients treated for epilepsy, both in adults (35,36,37,38) and children (39), show low serum concentrations of calcifediol, in the latter case with clinical manifestations of rickets (40,41). Theophyllamin also lowers the serum concentrations of calcifediol in rats treated for 4 weeks with this drug (42). In these two cases the cause is an induction of the liver's microsomal system.

Variation of the sunlight is reflected in the serum concentration of calcifediol. It is known from investigations in Europe (43,44,45,46), Japan (47), USA (48) and Antarctica (49) that the amount of 25-OH- D3 in the population varies with the season as a consequence of the seasonal variations in UV-radiation. Variations depending on latitude are demonstrated in a study from Argentina, where comparisons of concentrations of 25-hydroxyvitamin- D3 in two populations in Buenos Aires (34S) and Ushuaia (55S) in August (corresponding to late winter) showed a clear difference, 21.1 and 9.3 ng/ml respectively (50). North of the north polar circle the amount of sunlight is so small that there is no production of vitamin D during the winter (51). Lack of vitamin D synthesis in the winter has been demonstrated as far south as in Boston at a latitude of 42N ( 52). The studies mentioned so far all refer to man, but there are also investigations of sheep ( 53), and horses (54).

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Calcitriol - The hormonal form of vitamin D3

Calcifediol is in turn hydroxylated to calcitriol (1,25-(OH)2-D3). Two hormonal systems are involved: a parathyroid-hormone-sensitive system in the kidney's proximal tubuli (55,56) which has cAMP as a second messenger (57) and a system sensitive to calcitionin in the descending straight tubuli (58). Its second messenger is not yet determined. Calcitriol is the metabolically active and hormonal form of vitamin D3. Normally, the synthesis in the kidneys is the only one of systemic importance. Its serum concentration is dependent upon the kidney function and it is not measurable in individuals without kidneys (59,60,61,62,63). Cells in other tissues also have the possibility to produce calcitriol (64 65,66,67,68). First of all, granuloma tissue (69) as in sarcoidosis, which is the cause of the hypercalcemia in this disease (70,71), tuberculosis (72,73,74,75,76) and lepra (77,78). Production of calcitriol normally also occurs in the placenta (79). It is possible to induce synthesis of calcitriol in several other types of cell cultures (80,81,82,83,84) even if its physiological significance is unclear. Of special interest is the production if this hormone by different cells in the immune system (85).

In isolated liver tissue from the rat, calcitriol inhibits the production of calcifediol (86,87). It has been shown in several studies, in both animals and man, that the content of calcium in the food influences the serum concentration of calcifediol (88,89,90,91). More detailed investigations have revealed that this effect is mediated by parathyroid hormone (PTH). When the content of calcium in the gut is low, this hormone is stimulated to be able to increase the absorption of calcium in order to maintain normal blood concentrations of calcium. PTH stimulates the production of calcitriol and this in turn gives an increased breakdown of calcifediol. The exact mechanism for this is not determined.

15

Renal metabolism of vitamin D3

The calcitriol metabolism in the proximal tubules of the kidneys is tightly regulated. First of all this substance controls its own synthesis in a feed-back regulated loop (92,93,94). Serum phosphate also has a strong regulatory effect and is responsible for the switch in synthesis between calcitriol and 24,25-(OH)2-D3 (95). In thyro-parathyroectomised rats, low serum phosphate stimulates the synthesis of calcitriol (96) but if the phosphate values rise over 8 mg/100 ml the synthesis of calcitriol is shut down and the production of 24,25-(OH)2-D3 is stimulated. An intact hypopituary function is necessary for this regulation to take place (97), because hypophyseectomised rats need a supplement of growth hormone and triiodothyronine to allow this control by phosphate (98,99,100,101). The intake of phosphate in the food also controls the production of calcitriol in other animals, such as pigs (102) and man (103). Serum phosphate, on the other hand, is elevated by calcitriol by stimulation of the intestinal uptake (104,105) through stimulation of active transport in the vesicles in the brush border (106,107). This transport is independent of and different from the transport of calcium (108).

Calcitriol is a calcium elevating hormone. It increases the calcium uptake from the intestine (109,110,111). Sodium has a not fully clarified role here (112). It also stimulates the reabsorption of calcium in distal tubuli (113) and increases the mobilisation of calcium from the bones (114,115), by stimulating osteoclast activity (116).

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Calcium

Calcium by itself, also directly regulates the synthesis of calcitriol (117,118). In the rat, during constant infusion of PTH, the serum concentrations of calcitriol can be varied by changing the serum concentrations of calcium using EDTA, this without changes in serum phosphate (119). Experiments with PTH infusion in dogs and humans show that calcium can regulate the synthesis of calcitriol at high serum levels of parathyroid hormone (120). Whether this is a physiological regulatory mechanism is not clear. Calcium also modulates the effect of PTH on the 1hydroxylase in the rat (121).

Parathyroid hormone

Parathyroid hormone and calcitriol balance each other by a negative feed-back loop. PTH stimulates the synthesis of calcitriol which in turn suppresses PTH synthesis (122,123,124). This action is exerted directly on the synthesis of PTH (125,126,127) and indirectly by increasing the intracellular concentration of calcium in the parathyroid cells, thereby changing the sensitivity to serum calcium in these cells (128,129,130). Calcitriol also has an inhibitory effect on parathyroid cell proliferation (131)

Aluminium

Aluminium suppresses 1 hydroxylase in the proximal tubuli, both in subjects with normal kidney function (132) and in renal failure (133,134). Like calcium, uptake of aluminium from the gut is stimulated by calcitriol, both in the rat and in man

17

(135,136). This metal ion also inhibits the release of parathyroid hormone (137,138,139,140).

18

THE AIM OF THE STUDY

The aim of the study was to investigate the metabolism of vitamin D in relation to sun light, calcium, and phosphate, and answer the following specific questions:

Is there a correlation between vitamin D3 metabolites and sunlight at different latitudes? (I, II, and III)

Is there a connection between the calcium content in the diet and vitamin D3 metabolites and clinical signs of rickets? (IV)

Can control of serum phosphate by phosphate binders or diet affect the production of calcitriol? (V and VI)

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MATERIALS AND METHODS

Vitamin D in the Antarctic region

The first two papers, I and II, concern humans and animals from the Antarctic region.

Humans

Venous blood was also taken from 4 volunteers participating in the Norwegian 1991-92 "Aurora" expedition to Filchner Ice Shelf (75S). They were sampled before departure and after arrival in Montevideo, in December and March respectively.

Animals

Blood samples were collected from male gentoo penguins (Pygoscelis papua) at the beginning of November 1991 (early spring) (n=10) and beginning of April 1992 (autumn) (n=10) at a penguin rookery near Uruguay's Antarctic base "Artigas" on King George Island (6211'S, 5851'W), situated east of the Antarctic Peninsula. The blood was drawn from a superficial vein under the flipper and the sex was determined by measuring the bill using the method of Williams (141).

20

Samples of plasma from female elephant seals (Mirounga leonina), taken at Husvik, South Georgia (5410'S 3643'W) during the 1990-91 season were obtained through the British Antarctic Survey. The samples were taken at the end of the breeding period in November, just before they went to sea (n=8), at the beginning of moulting in January (n=5),and at the end of moulting in February (n=4).

Blood was sampled from the extradural vein. All blood samples were immediately transferred to heparinised tubes and, after separating the plasma, stored at -18C until analysed in Sweden.

In paper II, the investigations on the elephant seals were extended. The study was carried out at Husvik during September-November 1988 and October-February 1990-91. A total of 34 adult female southern elephant seals were individually marked with plastic rototags (Dalton Ltd., UK) in the web of the left hind flipper. Presence of individual seals was noted during daily counts. All females were tranquillised using a mixture of tiletamine hydrochloride and zolazepam (142) and weighed to the nearest kg in a net stretcher suspended from a load cell attached to an aluminium tripod (143). In September-October 1988, twelve females were tranquillised at early breeding (0-4 days after giving birth). All twelve females were recaptured at the end of breeding in October-November (0-5 days before weaning the pup). In October-November 1990, an additional nine females were tranquillised at the end of breeding (1-7 days before weaning the pup).

In January-February 1991, eleven females were tranquillised at early moulting (minimum of 0-4 days after arriving ashore). Seven of these were recaptured in

21

January-February together with three additional females at the end of the moulting (minimum of 0-2 days before departure to sea).

Daily rates of change in calcifediol and calcitriol during breeding and moulting were calculated using individual seals which were sampled both at the early and late stages of breeding (12 seals) and moulting (7 seals), respectively. The rates of change were calculated by subtracting the calcifediol and calcitriol levels in the early stages from the levels at late stages and dividing the difference by the number of days between the taking of these samples.

The levels of calcifediol and calcitriol on arrival to breed and moult were estimated for individual seals by extrapolating the average daily rate of change (measured over a period of at least 12 days) from the day of the first sampling occasion back to the day of arrival. The levels of these metabolites on departure (end of breeding or moulting) were estimated by extrapolating the calculated daily rate of change from the last sampling occasion to the day of departure.

Two different sets of data are presented; recaptured seals and pooled seals. Recaptured seals are individual seals which were captured in early stages and then recaptured in the late stages of breeding (12 seals) and moulting (7 seals), respectively. Pooled seals are the recaptured seals pooled with the seals which were sampled once.

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Clinical studies

A Swedish population

The third study was performed in association with the screening procedures for the WHO MONICA (MONitoring trends and determinants In CArdiovarscular diseases) Project Gteborg, Sweden (144). In 1984 1000 men and 1000 women aged between 25 and 64 years, selected at random from the population census of the city of Gteborg, were invited to participate in the first MONICA screening. Out of the invited 2000 subjects, 1421 (71%) participated. For the analysis of vitamin D3 metabolites, 50 samples from each age-group were selected at random. A few samples could not be used and altogether 184 men and 198 women were available for analysis. These blood samples were frozen and stored at -70C until analysis which was done within one year for all variables except for the hormones and osteocalcin, which were analysed after 7 years.

Children in Uruguay

The third study (III) was performed in collaboration with the Uruguayan Ministry of Public Health. During the period of August to December 1989, a field study was performed in "Las Villas de Las Piedras" (145), a low-income suburban area 24 km north of the capital Montevideo. A nested design was used to get a random population sample, of 163 children, 84 boys and 79 girls, born in 1984 or later, belonging to 104 families. In each child a clinical examination was made by the author. Signs of rickets detected by inspection and palpation and the following signs were noted: frontal bossing, rachitic rosary, Harrison's groove, and

23

enlargement of joints. At the same time a recording of the food intake during the 24 hours before the interview was made by an experienced nurse. The milk intake was estimated by measuring the volume of the drinking container the child usually used. In the case of some children, randomly elected, parental permission to drawing blood samples for the analysis of calcifediol and calcitriol was granted. Nine with signs and 7 without signs of rickets were sampled in this manner. The serum from these samples was kept frozen at -20 C until analysed in Sweden.

Renal patients

In Paper V and VI patients from the department of Nephrology at Sahlgrenska sjukhuset were studied.

In paper V nine patients were studied. They had end-stage renal disease of various causes and were treated with chronic maintenance hemodialysis. Time in hemodialysis varied between 5 and 82 months, mean 32.1 months. No patients were taking medications containing vitamin D. The investigation was divided into three parts, each lasting four weeks. During the first period, the phosphate binding dose was standardised to 0.5 g aluminium hydroxide tablets (Minicid), 2 tablets three times daily with meals. During the second period the treatment was discontinued so that the patients were without any phosphate binder. During the last period calcium carbonate was given three times a day together with meals in a dose of 2.5 g each, which corresponds to 1 g of elemental calcium. Serum levels of calcium and phosphate were measured at the end of each period. For the determination of these last two metabolites, the serum samples from all the periods were stored at -20C and analysed together at the end of the study. Paper VI concerns 16 patients who started a protein-restricted diet. Of these, 9 patients

24

had a diet restricted to 20 g protein per day and 7 received 40 g protein per day. There were no significant differences between the two groups in any clinical parameter and all the data from the two groups were therefore pooled. Patients treated with vitamin D or its analogue and patients visiting sunny countries during the study period were excluded. The measurements of the vitamin D-metabolites were repeated after 3 and 6 month's treatment.

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Analytical methods

Calcifediol

After extraction with acetonitrile and cleaning on a Sep-Pak C18 column (146), calcifediol was measured in serum with a protein binding assay with normal rat serum as a source of binding protein (43), Paper II and III. Later the method was replaced by a commercial direct radioimmunoassay without a clean-up step (INCSTAR, Stillwater, Minnesota, USA), Paper IV, V, and VI.

Calcitriol

In all studies in this thesis, calcitriol was measured with a radioreceptor assay with binding protein from calf thymus (147) from INCSTAR.

Parathyroid hormone (PTH)

PTH was measured by a two-step N-terminal immunosorbent method followed by a mid-region radioimmunoassay (PTH N-tact, Immuno Nuclear Corporation, Stillwater, Minnesota, USA), normal range 3-9.3 pM (148,149), Paper V.

Other chemical analyses

Serum concentrations of total calcium, phosphate, urea and creatinine were analysed at the Department of Clinical Chemistry at Sahlgrenska University

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Hospital (an accredited laboratory No 1240, according to EN 45001) by established methods. Glomerular filtration rate (GFR) was measured as 51CrEDTA clearance (150). Total cholesterol, high density lipoprotein, cholesterol and triglycerides were determined enzymatically (Boeringer Mannheim, Mannheim, Germany) (151,152,153). Plasma fibrinogen was analyzed according to a polymerization method originally described by von Claus (154). Serum IGF-1 was determined by a hydrochloric acid-ethanol extraction radioimmunoassay using authentic IGF-1 for labelling (Nichols Institute Diagnostics, San Juan Capistro, California, USA).

Statistical methods

The Statgraphics 2.0 statistical package was used for statistical calculations. For comparison between groups the Wilcoxon ranked sign test and Mann-Whitney Utest were used when appropriate. Multivariate regression and simple correlation and regression coefficients were used to correlate changes in variables.

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RESULTS

Correlation between vitamin D3 metabolites and sunlight at different latitudes

In Antarctica

In penguins there were a clear difference the serum concentrations of calcifediol. From 10.8 ng/ml (SD 4.01) in the spring it increased to 20.9 ng/ml (SD 8.04) in the autumn, p<0.01, Fig 1.

Figure 1

30 20
ng/ml

10 0
spring autumn

25- hydroxyvitamin- D3 plasma concentrations for gentoo penguins (Pygoscelis papua) in spring and autumn, p<0.01.

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The humans also showed an increase in 25-hydroxyvitamin-D3, from 30.2 ng/ml (SD 3.11) to 40.2 ng/ml (SD 6.55), p<0.05, Fig. 2.

Figure 2

50 40
ng/ml

30 20 10 0
December March

Calcifediol plasma concentrations in members of the "Aurora" expedition at the time of leaving and after returning to Montevideo, p<0.05.

For the seals, there were no differences in 1988 and 1990 late breeding levels of calcifediol and calcitriol. The 1988 and 1990 samples for late breeding were therefore pooled.

A summary of levels of calcifediol and calcitriol for both recaptured seals and the population for the four different stages is shown in Table I. There were no differences between the recaptured seals and populations in levels of calcifediol and calcitriol at any stage. There were, however, seasonal changes in both calcifediol and calcitriol (ANOVA, p<0.01 and p<0.05 respectively).

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Also, the decrease in calcifediol between late breeding and early moulting was significant (P>0.001). However, the increase of calcitriol between late breeding and early moulting was not significant at the 0.05 level.

Table I

Calcifediol (ng/ml) n Breeding Early Late Early Late 12 12 7 7 Recaptured 112 30 142 48 74 15 100 24 n 12 21 10 9 Pooled 112 30 129 42 74 13 112 40

Moulting

Calcitriol (pg/ml) n Breeding Early Late Early Late 8 8 7 7 Recaptured 102 30 88 32 100 30 77 18 n 10 9 11 10 Pooled 103 28 72 42 105 41 75 40

Moulting

Levels ( SD) of calcifediol and calcitriol in plasma in both early and late breeding and moulting periods in female southern elephant seals. The recaptured column represents individuals for which samples were taken both in the early and in the late stages. The pooled column represents all seals which were sampled in that specific time period, n= sample size, calcifediol p<0.01, calcitriol p<0.05 (ANOVA).

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There were significant increases in calcifediol level during both breeding and moulting (Table II). However, the decrease in calcitriol levels during breeding and moulting was not significant (Table II). There were no significant differences (P>0.1) when comparing the rate of increase in calcifediol and rate of decrease in calcitriol between breeding and moulting.

Table II

25-OH-D3 (ng/ml * d-1) Breeding Moulting 1.7 1.3 1.1 0.6

1,25-(OH)2-D3 (pg/ml * d-1)

12 7

<0.01 <0.01

-0.9 1.7 -1.1 1.6

8 7

NS NS

Daily rates ( SD) of change in serum of calcifediol and calcitriol during breeding and moulting periods in female southern elephant seals. Sample size = n. The paired t-test was used to analyze if the rates were different from 0.

Figure 3a and 3b show the levels for the population of calcifediol and calcitriol at the four stages. There were significant differences between the different stages in levels of both calcifediol and calcitriol (One-way Anova, ;calcifediol, P<0.01; and calcitriol, P<0.05).

31

Figures 3a and 3b

2 -O -D 3 5 H 20 0

10 5

n /m g l

10 0

5 0

0 O o ct/N v Ja /F b n e Ot c

Estimated seasonal changes during a year of a) calcifediol (p<0.01) and b)

1,25-(OH) -D 3 2 200

150

pg/m l

100

50

0 Oct/Nov Jan/Feb O ct

calcitriol (p<0.05) in female southern elephant seals. The vertical bars show the standard deviation. Note that the concentrations of calcifediol are a thousand times higher than the concentrations of calcitriol

The rate of increase of calcifediol and rate of decrease of calcitriol during breeding and moulting were regressed against each other. No significant

32

correlations were observed. There was no relationship in total mass loss of females and total change of calcifediol and calcitriol during breeding and moulting.

In a general Swedish population

Body height, weight, BMI, and waist to hip circumference ratio (WHR) for the different age-groups in men and women are given in table III. Body height decreased by 5 cm for men and 5.3 cm for women between ages 25-34 and 55-64 years. Body weight and BMI increased, whereas WHR increased only slightly with increasing age.

33

Table III

Men Age, years 25-34 35-44 45-54 55-64 All Women Age, years 25-34 35-44 45-54 55-64 All

n 48 49 47 49 193

Height cm 180 178 177 177 178

Weight kg 77.2 78.4 81.2 80.1 79.2

BMI kg/m2 23.8 24.6 25.9 25.7 25.0

WHR 0.92 0.93 0.95 0.93 0.93

49 49 50 43 191

167 166 165 163 165

60.6 64.7 64.8 68.3 64.5

21.8 23.5 23.9 25.5 23.6

0.80 0.81 0.80 0.83 0.81

Mean body height, weight, body mass index (BMI) and waist-to-hip ratio (WHR) by sex and age

Table IV gives values for calcifediol and calcitriol concentrations by sex and agegroup. calcifediol did not differ between the sexes but for men, it decreased significantly with increasing age. calcitriol was not related to age in bivariate analyses in either men or women.

Table IV

Men Age, years 25-34 35-44 45-54 55-64 All Women Age, years 25-34 35-44

n 47 47 47 49 190

25-OH-D3 ng/ml 39.9 35.4 35.0 30.2 35.2

SD 8.2 13.5 15.5 13.7 13.3

1,25-(OH)2-D3 pg/ml 25.5 25.5 30.6 25.2 26.7

SD 9.3 6.7 8.2 8.2 8.4

50 50

38.6 31.4

19.8 13.1

25.2 29.6

13.2 10.4

34

45-54 55-64 All

49 43 192

33.0 38.6 35.2

14.6 16.4 16.4

34.8 27.8 29.4

11.6 8.4 11.6

Concentrations of vitamin D metabolites by sex and age

Figure 4

50 40
pg/ml ng/ml

Cal 0Ca

30 20 10 0

10

11

Month Concentrations of calcifediol and calcitriol by month of the year. Values for calcifediol in months 6, 8 and 9 are significantly higher, p<0.01.

The time of the year is known to influence on circulating calcifediol concentrations and in figure 4 the seasonal variation for is shown both for calcifediol and calcitriol in relation to the month of the year. It will be seen that calcifediol concentrations were significantly higher during June, August, and September compared with all other months, whereas calcitriol did not vary significantly with the time of the year, figure 4. Out of all other variables studied, only systolic blood pressure was significantly lower during the sunny months

35

(123 mm Hg vs. 130 mm Hg; p<0.001) and WHR which was slightly higher during the sunny months (0.88 vs. 0.86; p<0.05).

In the bivariate correlation between calcifediol and calcitriol vs. all study variables, calcifediol correlated positively to physical activity in both men and women and negatively to intact PTH in both sexes, and also negatively to systolic and diastolic blood pressure in men, whereas no such correlation was seen in women. calcitriol was positively correlated to intact PTH in both sexes and negatively to body height in men and psychological stress and osteocalcin in women.

When age and sun exposure were taken into account in the multivariate analysis, calcifediol was only significantly (negatively) correlated to intact PTH, for men r= -0.24 and for women r= -0.18; p<0.05.

A multivariate regression analysis with calcitriol as the dependent variable showed a negative correlation with age in men and no correlation in women. However, when men and women were analysed together, there were independent correlations with age and positive ones with intact PTH and osteocalcin.

Is there a connection between the calcium content in the diet and vitamin D3 metabolites and clinical signs of rickets?

One or more signs of rickets were found in 64 (39.3%) of the children. These findings were especially common in children born in 1987, where 17 out of 31 (54.8%) presented at least one sign of rickets without significant differences between the sexes. Most of the clinical findings were mild ones, but 16% of the

36

children showed more advanced signs of the disease such as deformities of knee and/or wrist joints (figure 1, table IV). It should be observed that the development of signs showed a "cephalo-caudal" direction with relation to age, beginning with deformities of the head then, the thorax and finally the joints. There was relatively little overlapping between signs from the head and joints (fig. 5, Table V)

37

Table V

Age group 0 1 2 3 4 5 0-5

Total with/without signs of rickets (%) 1/19 (5) 15/15 (50) 17/14 (54.8) 11/13 (45.8) 12/25 (32.4) 8/13 (38.1) 64/99 (39.3)

head and/or (%) 0 8 (26.7) 8 (25.8) 4 (16.7) 4 (10.8) 2 (9.5)

Deformities of thorax and/or (%) 1 (5) 12 (40) 15 (48.4) 9 (37.5) 5 (13.5) 4 (19)

joints (%) 0 3 (10) 7 (22.6) 5 (20.8) 7 (18.9) 6 (28.6)

Distribution of cases with signs of rickets in different age-groups

Figure 5

50 40 r m 0 A (years) ge 1 2 3 H arrison grove 5 Frontal bossing R achitic rosary Y 4 T K nee W rists 30 20 10 0 % " $ & (

Signs of rickets in different age-groups

38

39

Figure 6

8 14 14 Thorax 3 15 1

Head

9 Joints

Overlapping of signs of rickets from different body locations

It was not possible to get exact data about the sun exposure of the children. The general impression was that the mothers tended to protect the smallest children (< 2 years) from the sun but could not watch the older children.

The average serum concentrations of calcifediol were 19.8 ng/ml (SD 3.55) and 26.6 ng/ml (SD 5.1, p<0.02) for the groups with and without signs of rickets respectively. In the case of calcitriol, the values were 46.8 pg/ml (SD 4.7) and 43.0 pg/ml (SD 4.3) (not significant). The regression of calcitriol on calcifediol was significant for the 7 children in the group with signs of rickets (correlation coefficient=-0.69, p<0.02), fig. 7.

40

Figure 7

Calcitriol (pg/ml) 70

60

50

40

30 10 15 20 Calcifediol (ng/ml) 25 30

Regression of calcitriol on calcifediol in children with signs of rickets, r=-0.69, p<0.02

A general finding in the food registration was that the population was very poor and nearly all the families had economic difficulties purchasing what is considered in the western world to be an acceptable and varied nutritional supply. Cow milk was consumed by all the children except for 4 breast-fed infants as well as by some of the children who were partially breast-fed (born during 1989). Much of this milk was distributed free by the National Institute of Nutrition in Uruguay (INDA) or by the church and other help organisations. This was found to be practically the only source of calcium. Other potential calcium sources such as cheese and other milk products or pulses and vegetables were consumed in very small quantities.

41

Because of the breast-feeding, the 20 children born during 1989 were excluded from the calculations below.

A significant difference in milk consumption was found between the group with clinical signs of rickets and the group without such signs (p<0.03). A comparison of these two groups in each age-group showed a significant difference for the 4 and 5-year-olds (4 years old p<0.01 and 5 years old p<0.04), fig. 4. It was also found that the older children ingested less milk than the younger children in both of these groups (rickets p<0.002, no rickets p<0.04).

Control of serum phosphate by phosphate binders and the production of calcitriol

In hemodialysis patients, replacement of the aluminium-containing phosphate binder by calcium carbonate increased serum calcium and calcitriol and decreased PTH whereas phosphate and the calcium-phosphate product showed no significant difference with the two treatments. During the interposed second period both phosphate and calcium increased. Five of the nine patients had episodes of mild hypercalcemia (serum calcium between 2.7 and 3.0 mmol/l) during treatment with calcium carbonate. Serum calcium was normalised by dose reduction of the calcium salt without changes in the levels of serum phosphate.

42

TABLE VI

Treatment period

Ca (mM) 2.27 0.17 2.38 0.21 2.57 0.10 p<0.05 p<0.01 p<0.05

P (mM) 1.51 0.50 2.26 0.50 1.63 0.48 p<0.01 NS p<0.05

Ca x P

PTH (pM) 22.4 26.8 24.8 26.9 16.4 24.2 NS p<0.05 p<0.05

Calcitriol (pg/ml) 8.0 3.3 8.3 4.8 11.5 4.8 NS p<0.05 p<0.05

1 (Al(OH)3) 2 (no binder) 3 (CaCO3) 1 vs. 2 1 vs. 3 2 vs. 3

3.44 1.11 5.38 1.38 4.19 1.32 p<0.01 NS NS

Serum concentrations SD of calcium (Ca), phosphate (P), calcium- phosphate product (Ca x P), PTH and calcitriol at the end of each study period and tests of significance of differences between these periods.

In patients on protein-restricted diet the renal function, measured as 51Cr-EDTA clearance and serum creatinine, deteriorated only slowly during the study. Serum urea levels fell significantly after the initiation of the protein-reduced diet and remained stable during the time of investigation (table VII). There were no changes of the mean body weight. Calcitriol levels rose significantly (p<0.05) after 3 months from 17.1 pg/ml to 27.7 pg/ml but fell after another 3 months to almost their initial value, 15.3 pg/ml. The serum phosphate levels decreased during the same periods from 1.99 to 1.67 to 1.93 mmol

43

Table VII

Ca (mM) at start 3 months 6 months start vs. 3 months start vs. 6 months 3 months vs. 6 months 2.23 0.22 2.35 0.18 2.31 0.22

P (mM) 1.99 0.40 1.67 0.38 1.93 0.55

GFR (ml/min) 8.3 4.7 8.5 4.8 7.9 5.9

Creat (M) 550 180 572 189 615 214

Urea (mM) 27.1 8.1 22.7 8.9 22.9 8.7

StB (mM) 23.4 2.8 23.8 2.3 22.6 2.0

1,25 (pg/ml) 17.1 10.5 27.7 20.9 15.3 8.3

25-OH (ng/ml) 28.1 14.3 29.1 16.8 33.7 17.7

p<0.01 NS NS

p<0.01 NS NS

NS NS p<0.05

NS p<0.01 p<0.05

p<0.05 p<0.05 NS

NS NS p<0.05

p<0.05 NS p<0.05

NS NS NS

Renal function, serum urea, standard bicarbonate, calcium, phosphate, calcitriol, and calcifediol concentrations (mean and SD) at the start, at 3 months, and after 6 months with significancies. Ca=calcium, P=phosphate, GFR=glomerular filtration rate, Creat=creatinine, StB=standard bicarbonate, 1,25=calcitriol, 25-OH=calcifediol.

There was a negative correlation between the calcitriol and phosphate levels at the start and after 3 months but not after 6 months. There was also a significant correlation between the changes in calcitriol (-1,25) and initial GFR (Fig. 8). A breakdown of the results after 3 months into patients with increased calcitriol concentrations and patients with decreased or unchanged concentrations showed that the latter group had significantly higher phosphate and creatinine concentrations and lower standard bicarbonate concentrations (table VIII)

Figure 8

delta-Calcitriol (pg/ml) 40 30 20 10 0 -10 0 5 10 15 20 25 GFR (ml/min)

Relationship between the difference between calcitriol concentrations at the start and after 3 months and GFR. (Y=-2.02+1.54X, r=0.50, p<0,05)

45

Table VIII

-1,25

negative or zero 5 2.3 0.21 2.0 0.28 30.2 12.3 7.3 2.8 729 201 30.2 12.3 21.8 1.3 34.5 17.2 9.6 4.5

positive

significance

No of patients Ca P Urea GFR Creat Urea StB 25-OH 1,25

11 2.4 0.16 1.5 0.28 25.7 5.6 9.1 5.5 500 141 25.7 5.6 24.7 2.1 26.6 17.0 36.0 20.2 NS p<0.01 NS NS p<0.05 NS p<0.05 NS p<0.01

Renal function, serum urea, standard bicarbonate, calcium, phosphorous, calcitriol, and calcifediol concentrations (mean and SD) after 3 months in patients with decreasing (-1,25 negative or zero) and increasing (-1,25 positive) calcitriol concentrations. Ca=calcium, P=phosphate, GFR=glomerular filtration rate, Creat=creatinine, StB=standard bicarbonate, 25-OH=calcifediol, 1,25=calcitriol.

Another comparison was made between patients who had calcitriol concentrations greater and less than 20 pg/ml after 3 months. No significant differences were found, but phosphate and creatinine tended (0.1>p>0.05) to be lower and standard bicarbonate, GFR and body-mass index to be higher in the group with calcitriol concentrations above 20 pg/ml. Further analysis by multivariate regression with stepwise variable selection with calcitriol as the dependent variable and phosphate, calcium, renal clearance, urea, standard bicarbonate, calcifediol and body-mass-index

46

as independent variables was performed to select those to include in a linear model. This was done for each period and for the pooled set of data. Each separate period did not show significant values for any variable but for the pooled set significant values were obtained, with positive coefficients for GFR (p<0.0002, coeff=1.44) and BMI (p<0.0002, coeff=1.16) and a negative coefficient for serum phosphate concentrations (p<0.004, coeff=-10.24), R2=0.77 for the full regression.

47

DISCUSSION

Sunlight and calcifediol

Vitamin D3 metabolites have not previously been measured in a systematic way in animals and man in Antarctica. Because of the wasteness of Antarctica and research priorities earlier studies of vitamin D3 on this continent are confined to sporadic observations which are not so easy to repeat (49,155).

In penguins in Antarctica

The study by Griffiths and Fairney (49) did not show any seasonal difference in 2 species of penguins (P. adelie and P. antarctica). However, since our study had a different sampling interval and investigated another species in a different geographical location, our results are not comparable.

In humans in Antarctica

In man a clear seasonal variation has been demonstrated. The present data supports these earlier findings.(49) During a stay of 10 weeks on the Antarctic continent, the UV-B radiation on exposed skin is sufficient to elevate 25-hydroxyvitamin-D3 concentrations in plasma significantly.

48

In elephant seals in Antarctica

The increased levels of calcifediol in female southern elephant seals, during breeding and moulting, follow the general pattern for mammals which are exposed to sun light. Our study shows that female southern elephant seals have two different peaks of calcifediol. This has not been observed in any other animals, which have only one peak per year, around early autumn (44). For female southern elephant seals, the two peaks coincide with the periods spent on land during breeding (October-November) and moulting (January-February). At sea, elephant seals spend about 90% of their time under water (156,157,158) and it is probable that only the head is above the surface while at sea. The time ashore during breeding and moulting is the time when the most of the annual D vitamin synthesis occurs. The daily rate was the same during the two periods on land.

During lactation there is an increased demand for calcium. The calcium is transferred to the pup through the mother's milk. A rough estimate of the total amount of calcium transferred from the mother to her pup during lactation is 65 g (159,160,161). Female northern elephant seals (Mirounga angustirostris) are estimated to transfer 10% of their skeletal mass to their pups during the lactation period (162). After weaning, females spend 70 days at sea (163) when the calcium deposits in the bones must be replenished. The demand for calcium is probably higher during lactation than during moulting. The decrease, at sea, of calcifediol after breeding may be an effect of the restoration of calcium deposits. To meet this increased demand for calcium, the blood parathyroid hormone (PTH) may be increased. This elevated level of PTH stimulates the production of calcitriol, which inhibits the production of calcifediol and also stimulates the break down of this substance (89). The estimated levels of calcifediol in late moulting and early breeding were the same (fig. 9), despite the fact that the seals had been to sea for about 240 days. This may indicate that females at the end of moulting do not have a negative calcium balance as seems to be the case after the 49

breeding period. In northern elephant seal females bone resorption predominates during the lactation while there is bone formation during the moult (162).

The only other study of calcifediol levels in seals was performed by Griffiths and Fairney in 1988 (49). They showed a difference in calcifediol levels between the crabeater seal (Lobodon carcinophagus) (21 ng/ml) and the weddell seal (Leptonychotes weddelli) (64 ng/ml). They concluded that the difference in calcifediol levels could be explained by the diet of the seals. A confounding factor was that the crabeater seals were mainly sampled during the winter and the weddell seals during the summer months. Crabeater seals feed almost entirely on krill (Euphasia suberba) (164), which probably contains very little vitamin D like other shellfish ( 165), while weddell seals feed largely on fish (166), which contain high levels of vitamin D in their liver (165). The southern elephant seal feeds to a large extent on squid (167,168), which has vitamin D levels between shellfish and fish (169). Southern elephant seals do feed on fish, but to what degree is not known (170,171). The diet of southern elephant seals would suggest that they should have a level of calcifediol which is between the crabeater seal and the weddell seal. However, if we only take the prey into account as an indicator of the level of calcifediol in the southern elephant seals, the measured levels in this study were much higher than expected. The reason for this is not known.

In seals, annual layers of incompletely calcified and fully calcified dentine in teeth is used for aging (172). The mechanism for the variation of calcification of dentine is not known. It has been suggested that hormones such as testosterone and progesterone during breeding, and thyroxine during moulting, may affect the calcification (159,173). The southern elephant seal deposits four different layers of dentine per year (159,173) while other seals usually deposit only two (174). This would support Laws (159), who pointed out that higher D-vitamin levels in seals when on land than at sea could affect the deposition of calcium in their teeth.

50

During the last few years there has been a search for indicators of UV radiation. In moss samples it has been possible to use flavonoids (175) but this is an application limited to a group of plants. Another interesting approach has been the use of dosimeters with spores from Bacillus subtilis (176,177).

The diminution of the stratospheric ozone layer has alarmed governments all over the world. Since there has been a trend of decreasing ozone over the northern hemisphere for the period 1969-1986, and signs of an increase for the longer period 1957-1986, there is a need for more measurements (178) and methods to estimate UV-radiation (179).

These studies in Antarctica show the possibility that the vitamin D3 metabolite calcifediol can be a complement for assessing UV exposure in animals and man.

A general population in Sweden

The concentration of calcifediol in serum was similar in men and women. It was influenced by age in men only. There was a strong correlation between calcifediol concentration and the sunny season in the present study and this correlation was independent of other factors. This has been reported earlier in younger (180) and wellnourished, highly selected Americans (48), as well as in a Swiss ( 181) and a Danish population (182). There was no seasonal variation in calcitriol. Chesney et al (180) reported similar findings in young (18 months - 35 year old) individuals in the U.S. These authors explained the seasonal variation in calcifediol but constant calcitriol by a tight feedback regulation of the latter (180).

Interestingly, the present data showed a positive correlation between calcifediol and leisure-time physical activity in a multivariate analysis when age was adjusted for but

51

not when sun exposure was taken into consideration. Physical activities are often associated with being outdoors, and active individuals should therefore have a better chance of having sun exposure. Leisure-time physical activity was recently found to predict bone density independent of age and BMI in women aged 18-81 years (183). Thus, it cannot be excluded that the influence of sunlight on calcifediol while being outdoors might contribute to the increased bone mass in the previous report (183). We did not find any correlation between calcifediol and body weight. Bell et al reported in 1985 that obese subjects had lower concentrations of calcifediol (184). However, in that study, the subjects had higher calcitriol as well, but they also had a substantially wider weight range, with more obese subjects (184).

BMI correlated positively to intact PTH and calcitriol, respectively, and negatively to calcifediol (185). Furthermore, life style factors such as smoking and coffee consumption adversely influenced on PTH concentrations and hence, indirectly on vitamin D metabolism. The result of the multivariate analysis regarding calcitriol from the same population study (Paper IV) showed no sex difference. Age, which had no significant importance in the bivariate analysis was negatively correlated to calcitriol. This is in accordance with some other studies (186,187,188,189,190,) while some have reported no age correlation for calcitriol (48,181,182,191,192). In one of the latter studies with highly selected, healthy volunteers no relation between glomerular filtration rate and calcitriol was found (48). Thus, any relationship to age does not seem to be related to a decreasing glomerular filtration rate.

Rickets, calcium and the metabolism of calcifediol

There are only a few community surveys of rickets in the literature; most data are from populations in clinical settings. In these different surveys the prevalence varies greatly, from less than 1% to 60% or more [193,194]. The selection criteria also vary.

52

From this earlier experience, it can be concluded that the diagnosis of rickets by physical examination only is difficult and for a more definite diagnosis x-ray examination and/or laboratory data are necessary, which was difficult to achieve in our study due to its character of a field survey. It could therefore be expected that both false positive and false negative results would be encountered. However, our data are within the range of prevalences from earlier studies and once more underline the fact that rickets is not uncommon in a subtropic country.

The serum concentrations of calcifediol were significantly lower in children with clinical signs of rickets, though none of them were under the lower normal limit (15 ng/ml). It should be pointed out that this lower normal limit is based on the statistical distribution of values and not on the occurrence of disease. It is possible that mild signs of rickets can occur in the lower "normal" range of calcifediol serum concentrations.

Since milk was found to be the dominant form of calcium intake the, milk consumption was used as an index of calcium intake. Sporadic cases of calcium deficiency as a cause of rickets have been reported in the literature ( 195,196,197,198,199). When blood analysis has been available, the values for PTH have been high, calcifediol low or low in the normal range, and calcitriol high.

Recent animal research has shown that the half-life for calcifediol is related to the content of calcium in the food (89,200), animals with a low intake of calcium having a high breakdown rate of calcifediol and as a consequence lower concentrations of this metabolite. In human experiments, too, it has been shown that the concentration of calcifediol is related to the ingested calcium and inversely related to the serum levels of calcitriol(91). There is also information from clinical studies of primary hyperparathyroidism, which sometimes can present with symptoms and signs of osteomalacia, low levels of calcifediol and high levels of calcitriol (201). This has also 53

been observed in patients with sickle cell anaemia, where low serum calcium, high PTH and low calcifediol were found (202).

In our study, we found a significant linear correlation between the concentrations of calcifediol and calcitriol in the group with signs of rickets but not so in the group without such signs.

All these findings suggest the following sequence of events: a low nutritional calcium intake induces high levels of PTH, which has a direct stimulating effect on the synthesis of calcitriol in the kidney. PTH also inhibits the tubular reabsorption of phosphate in the kidney with lowered serum phosphate as a consequence and this also stimulates the renal production of calcitriol. These elevated concentrations of calcitriol induce an enhanced hepatic inactivation of calcifediol and because of these lowered levels of this metabolite symptoms and signs of rickets or osteomalacia appear.

This can explain the incidence of rickets in sunny countries by the following hypothesis: the solar radiation in these countries is sufficient for the production of vitamin D in the skin but if the children have an insufficient intake of calcium the turnover of calcifediol is accelerated and they enter a state of vitamin D-deficiency. From this, the clinical picture of rickets develops.

For cultural reasons mothers do not allow their children to be directly exposed to sunlight. This is true especially for smaller children and may explain the high incidence of rickets seen in the group of two-year-old children. The older children should have sufficient sun exposure to produce enough vitamin D to meet the body's requirements. However, these children had a low calcium intake and presented signs of more advanced rickets, taking more time to develop. This could well be explained by the theory presented above. 54

Control of calcitriol synthesis in renal failure

Inorganic phosphate (96) and calcium (119) are important regulating factors for calcitriol where high levels of both these compounds inhibit calcitriol synthesis. The rise in serum calcitriol when calcium carbonate was used as a phosphate binder was small and the significance was modest but this raises the possibility that phosphate is a regulator of calcitriol synthesis also in dialysis patients. This rise in serum calcitriol was not achieved by reduction of phosphate with aluminium hydroxide. The explanation for this is not clear but it has been reported from animal experiments that aluminium inhibits the release of calcitriol both directly (133) and indirectly by inhibiting the stimulating effect of PTH (203).

The lowering of PTH after calcium carbonate administration is presumably related to the rise in serum calcium per se.

An unexpected finding was the rise in serum calcium at the end of period 2. As phosphate rose, calcium also rose. But aluminium hydroxide is a binder of calcium as well as phosphate.

55

Calcium and phosphate are mainly absorbed in the small intestine. Their relationship can be described by the following equilibrium formula: Ca 3 PO 4

b g 3Ca
2

+ 2 PO 4

(1)

If the concentration of one component on the left side increases the concentration of the other must be reduced as a result of increased formation of calcium-phosphate complexes (204).

With addition of aluminium and hydroxide ions (OH-), the latter from the water in the gut, we get this formula: Ca 3 PO 4

b g+ Alb g+ OH 2Ca b g+ Al PO OH OH
2 3 2

+ Ca 2+ + PO 4

(2)

A comparison of (1) and (2) shows that the concentration of free phosphate ions will be the half and the amount of free calcium ions decrease by two thirds.

From this follow that the discontinuation of aluminium hydroxide should leave more calcium available for resorption and this could influence the serum levels of calcium.

To control hyperparathyroidism, the logical starting-point is to lower phosphate in blood with phosphate binders in the gut. The oral 1-hydroxylated vitamin D3preparations that have been proposed as a treatment to suppress PTH production give a first pass effect on the enterocytes with enhanced absorption of both phosphate and calcium. If calcium is brought into the body by a high dialysate calcium, its phosphate-binding capacity is not used. In both these treatments, there would still be a need for a phosphate binder. From this point of view hypercalcemia during calcium carbonate treatment should be corrected by lowering the dialysate calcium rather then by reducing the oral dose of calcium carbonate.

56

The interaction between calcitriol and phosphate has recently been regarded as the pivotal point in the development of the secondary hyperparathyroidism in renal failure. Instead of the earlier concept of a direct physico-chemical interaction between calcium and phosphate, the mechanism is now thought to be via the synthesis of calcitriol: high phosphate levels down-regulate the production of calcitriol and this in turn lowers the uptake of calcium from the alimentary tract and results in fall of the serum calcium levels (205).

When the protein in the diet was reduced, the calcitriol levels rose after the first 3 months but after 6 months they had returned to the initial levels. Because the phosphate levels moved in a reciprocal manner in comparison with calcitriol in the first two periods and there was a significant negative correlation between these two factors it can be conjectured that the lowered serum phosphate stimulated the 1hydroxylase in the kidney. The patients who showed a rise in calcitriol had better renal function after the first 3 months as measured with creatinine and better control of the metabolic acidosis. Later, however, the progression of uraemia made it impossible to control phosphate effectively by dietary means and other inhibiting factors of the hydroxylase may have been brought into play. Probably because of these other factors, the correlation between phosphate and calcitriol was lost after 6 months.

Furthermore, the change in calcitriol during the first three months was positively correlated with the initial GFR but in some of the most uremic patients with GFR lower than 10 ml/min there was even a fall in calcitriol levels, presumably because the hyperphosphatemia was not controlled in these cases. This is supported by the finding of Lucas et. al. that with severe renal failure (GFR <10 ml/min), after initiation of a protein-reduced diet, there is a fall in calcitriol concentration (8).

57

The multivariate analysis shows the importance of phosphate and renal function for the regulation of calcitriol synthesis and, rather surprisingly, body mass. The explanation for this relationship is not elucidated but it could be due to the fact that subjects with greater body mass have higher insulin levels and insulin stimulates calcitriol production (206). This study shows that there is a complex interaction of different factors which control the serum levels of calcitriol during protein restriction in uremic patients. From a clinical point of view, however, some conclusions can be drawn. The main determinant of calcitriol concentration in serum is the amount of functioning nephrons, i.e. renal mass, and the most important modulating factors which can be controlled by protein-reduced diet are serum phosphate concentrations and metabolic acidosis. Further clinical studies are needed to explore the therapeutic potential of these factors in the treatment of the disturbances in the calcium-phosphate metabolism in uraemia.

58

Notes on future research

The sun is the origin and only source of all life on this planet. For all time the sun light has been taken for granted and unchangeable. However, human action on earth has changed this during the last decades. The ultraviolet climate is changing due to a reduced thickness of the stratospheric ozone layer. Even if all activities contributing to this thinning were stopped now the natural balance will not be restored until the middle of the next century.

The spectral weighting, or biological "action spectrum" determines whether an incremental change in ozone results in significant changes in UV radiation that can be effective for biological photoreactions. To assess accurately the overall impact on the health of animals and humans quantitative analyses are needed of UV radiation effects on sensitive mechanisms which regulate the health of organisms.

To obtain a detailed knowledge of the changed UV climate at reduced ozone concentrations with its possible consequences on the biosphere, systematical biological UV-monitoring will be necessary. For humans, the comparable relationships can be developed either indirectly by comparing functions from animal data using epidemiological approaches or directly, where possible, by determining the dose-effect relationships such as that for the skin's production of vitamin D.

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SUMMARY

The overall aim of this study was to investigate the metabolism of vitamin D in relation to calcium, phosphate and ultraviolet radiation.

Vitamin D3 is formed in man and animals by radiation of the basal layer in the epidermis or the body surface with sun light in the ultraviolet range with wavelengths between 290 and 315 nm. In the liver, this vitamin is converted to calcifediol (25-OH-D3). Calcifediol is the vitamin D3 metabolite with the highest serum concentration (15-60 ng/ml). It has been shown in several studies, in both animals and man, that the content of calcium in the food influences the serum concentration of calcifediol. It is also the metabolite that shows the greatest seasonal variation. It is known from investigations in Europe, Japan, USA and Antarctica that the amount of this vitamin D metabolite in the population varies with the season as a consequence of the seasonal variations in UV-radiation.

Calcifediol is in turn hydroxylated to calcitriol. This is the metabolically active and hormonal form of vitamin D3. Normally, the synthesis in the kidneys is the only one of systemic importance. Its serum concentration is dependent on the kidney function and it is not measurable in individuals without kidneys. Serum phosphate also has a strong regulatory influence on the synthesis of calcitriol.

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The basic question was divided into the following specific questions:

Is there a correlation between vitamin D3 metabolites and sunlight at different latitudes? (I, II and III)

In a Swedish normal population, there is a strong correlation between calcifediol serum levels and the sunny season, independent of other factors. In Antarctica during the summer, the plasma levels of calcifediol increased in both penguins (Pygoscelis papua) and humans in spite of the high latitude. Female southern elephant seals (Mirounga leonina) in Antarctica also increased in their plasma levels of calcifediol significantly when they spent their time on land for breeding or moulting. No significant changes occurred in the levels of calcitriol during the same time.

Is there a connection between the calcium content in the diet and vitamin D3 metabolites and clinical signs of rickets? (IV)

Signs of rickets are relatively common (39.3%) in 0-5 years-old-children from a lowincome, suburban population in Uruguay. The children with rickets had lower serum concentrations of calcifediol, but still within the normal range. The intake of milk (and calcium) is lower in children 4 and 5 years old with signs of rickets.

Can control of serum phosphate by phosphate binders or diet affect the production of calcitriol? (V and VI)

In patients treated with hemodialysis, calcium carbonate is an alternative to aluminium hydroxide as a binder of the alimentary phosphate. The synthesis of calcitriol in these patients could still be increased by reduction of serum phosphate using calcium carbonate but not aluminium hydroxide. A protein-reduced diet in

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patients with renal failure also lowers the serum phosphate and increases the serum levels of calcitriol after 3 months of treatment but not after 6 months.

CONCLUSIONS

Sunlight is the most important determinant of the serum concentrations of calcifediol but not calcitriol.

Calcifediol shows a seasonal variation both in Sweden and in Antarctica and may be used as an index of UV-radiation.

Children with clinical signs of rickets have lower serum concentrations of calcifediol and a lower consumption of milk (and calcium).

Patients with end-stage renal disease, treated with protein-reduced diet or hemodialysis, still have a production of calcitriol that can be stimulated by lowering serum phosphate.

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ACKNOWLEDGMENT

This investigation is not the work of a single person, many have given me help to bring this work to its completion. Many sincere thanks to:

Mattias Aurell, head of the department of Nephrology and my tutor, for unfailing support, whatever I wanted to do.

Lisbeth Selvn, Inger Svensson, Lena Fors, Inga-Britt Petterson, and Miriam Fredlund at the research laboratory of the Department of Nephrology for the all important help with method development and analysis of vitamin D metabolites.

Mikael Sturn for introducing me to the methods of vitamin D analysis.

Eva Alopeus and all her staff, for expert library service, whatever the subject.

Krister Delin, Per-Ola Attman, Staffan Bjrk, Kerstin Landin-Wilhelmsen and Tom Arnbom for patience and helpful advice.

Gunilla Nilsson for keeping track of correspondence and research funds.

All community agents and doctors at the "Centro de atencin primaria para la sald", Las Piedras, Uruguay.

Nils-Holger Areskog, Anders Karlqvist, Archmedes Maciel, and Bartolom Grillo for helping me in my plans to go to Antarctica.

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Monica Kristensen and the Uruguayan Antarctic base commanders for help in obtaining blood samples from humans and penguins.

Special thanks to the station commander at the Russian base "Bellinghausen" the late Vladimir K. Stepanov who introduced me to the way of life in Antarctica.

Stefan Wikmark for putting his home to my disposal during my stays in Sweden.

Monika Puskeppeleit for support and help in bringing this work to an end.

Financial support has been given by the Faculty of Medicine of the University of Gteborg, Gteborgs Lkarsllskap, Swedish Society for Nephrology, Swedish agency for research cooperation with developing countries (SAREC), and Stiftelsen Lars Hiertas Minne.

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