16.6: Biomarkers of vitamin D status (18b.6)

Historically, vitamin D status was assessed indirectly by meas­uring alkaline phos­phat­ase activ­ity, as well as calcium and phos­phorus con­cent­rations in serum: all very non­specific indices. Methods are now avail­able for the direct measure­ment of vitamin D meta­bolites in serum, and these are des­cribed below. If possible, these measure­ments should be performed in con­junc­tion with an assay of serum para­thyroid hor­mone and some func­tional assess­ment of skel­etal health. In adults, this assess­ment may include measure­ment of bone mineral con­tent or bone mineral density. In chil­dren, in extreme cases of rickets, bony deform­ities such as en­larged fontanelle, rachitic rosary, and swollen joints are clin­ical signs of rickets, whereas knock knees or bowed legs are clin­ical signs of the assoc­iated osteo­malacia noted in growing chil­dren (Uday and Högler, 2019).

18b.6.1 Serum 25‑hydroxy­vitamin D

Serum 25‑hydroxy­vitamin D is the circulating meta­bolite of vitamin D that is the most abundant and has the longest half-life of all the vitamin D derivatives. Concentrations of 25(OH)D in serum (or plasma) are also the most useful measure of vitamin D exposure and status in humans, as they reflect the total supply of vitamin D from both cutaneous syn­thesis and diet­ary intake of either vitamin D₂ or vitamin D₃ (Wacker and Holick, 2013).Moreover, they can be used to define vitamin D de­fi­ciency, insufficiency, hypovitaminosis, sufficiency, and toxicity (IOM, 2011). Concentrations in healthy adults vary from 30–130nmol/L, depend­ing in part on expo­sure to solar ultra­violet light.

18b.6.2 Factors affect­ing serum 25 hydroxy­vitamin D

Seasonal and latitude effects on serum 25 hydroxy­vitamin D status are marked in many areas of the world. Subjects living north of latitude 33°N and south of latitude 33°S (Wacker and Holick, 2013) have reduced dermal vitamin D syn­thesis during winter, and show the highest serum 25(OH)D levels in the late summer, and the lowest in late winter. In a U.K. nat­ional survey of people > 65y (Finch et al., 1998), mean plasma 25(OH)D con­cent­rations were sig­nif­icantly higher in free-living participants surveyed in the summer (July–September) than in the winter (Figure 18b.3). Finch et al. (1998) further noted that in the summer, only 6% of a free-living group of elderly sub­jects had plasma 25(OH)D con­cent­rations < 25nmol/L com­pared to 35% of those who were insti­tutionalized.

Figure 18b.3. Comparison of mean plasma 25(OH)D levels by season for free-living elderly men and women and participants in institutions. Data from Finch et al., 1998, National Diet and Nutrition Survey: People Aged 65 Years or Over. The Stationery Office, London.

Only UVB rays (290–315nm) elicit syn­thesis of vitamin D₃ (chole­cal­ciferol). When the incident angle of the sun is low, UVB does not reach the earth (Grant et al., 2016). The “Shadow Rule” states that if one's height is longer than the length of one's shadow, vitamin D syn­thesis is possible; if not, the sun's incident angle is too low to provide UVB. This also explains why the best time for vitamin D syn­thesis is bet­ween 10:00 and 14:00 in summer in temperate countries or year-round in low latitude regions. Other factors affect­ing dermal syn­thesis are des­cribed below.

Age-related changes from infancy to adulthood in the con­cent­rations of serum 25(OH)D can be marked. Newborn infants have serum con­cent­rations that correlate with maternal 25(OH)D con­cent­rations. Breastfed infants, as noted above, are at risk of poor vitamin D status unless mothers have an adequate vitamin D status and can pass on meta­bolites in their milk (Stoutjesdijk et al., 2017). Vitamin D meta­bolites are lower when meas­ured in cord blood. Serum 25(OH)D levels in chil­dren of both sexes dec­line with increasing age ( Gregory et al., 2000), a trend that may reflect both behavioral factors (diet and sun expo­sure) as well as bio­logi­cal effects (increasing requirements of vitamin D with growth).

Studies indicate that calcium absorption by premature infants is not only vitamin D depen­dent; how­ever, concern remains about the lack of data to outline the vitamin D needs for premature infants (Taylor et al., 2019).

Older adults are part­ic­ularly vulnerable to low levels of serum 25(OH)D (McKenna et al., 1985). Lifestyle factors that reduce vitamin D status include low diet­ary intakes of vitamin D and limited sun expo­sure with a greater use of sunscreens and umbrellas. In addit­ion, there are bio­logi­cal reasons for the low status includ­ing a red­uced capacity of the skin to pro­duce vitamin D resulting from a reduc­tion of 7-dehydrochol­esterol in the skin, and impaired in­test­inal absorption of ingested vitamin D (Wacker and Holick, 2013). In coun­tries such as Canada where low vitamin D status is related to osteoporosis, it is recom­mended that older adults take vitamin D sup­ple­ments and age-related dec­lines in vitamin D status as meas­ured by serum 25(OH)D have not been observed in Canadian nat­ional survey data (Brooks et al., 2017).

Sex and Gender differ­ences in con­cent­rations of serum 25(OH)D have been noted, although no con­sis­tent pattern has emerged from nat­ional survey data in the USA (Wacker and Holick, 2013) and Canada (Brooks et al., 2017). Differ­entiating bio­logi­cal (sex) effects from lifestyle effects (gender)such as differences in food prefer­ences, clothing, and amounts of sun exposure during work or leisure may be difficult. In some coun­tries, consumption of fort­ified foods and sup­ple­ments as well as sunscreen use and sun avoidance practices, differ bet­ween males and females of all ages.

Skin pig­ment­ation differ­ences, often seen when comparing ethnic or racial groups, greatly influ­ences serum 25(OH)D con­cent­rations. Skin pig­ment­ation reflects the amount of the pigment melanin that absorbs UVB rays and lowers the amount of UVB acting on pre­vit­amin D. In the USA, for exam­ple, serum 25(OH)D con­cent­rations in African-Americans and Hispanics are much lower than in non-Hispanic whites (2011). In European coun­tries, African immigrants now living in northern latitudes have a greater risk of vitamin D de­fi­ciency com­pared to non-migrants. As an example, of the patients with nutri­tional rickets in Denmark, 74% were immigrant chil­dren (Beck-Nielsen et al., 2009). Not all differ­ences in vitamin D status bet­ween ethnic groups are due to skin pig­ment­ation. Behavioral differ­ences such as diet­ary intakes, clothing prefer­ences, and sun avoidance practices are also import­ant factors.

Melanin in skin does not block all chole­cal­ciferol syn­thesis, but it is slowed. Webb and Engelsen (2006) have cal­cu­lated the time needed for a person with each of the differ­ent Fitzpatrick categories of skin type to burn with sun expo­sure. Also shown in Table 18b.1, are the times needed to synthesize 1000 IU of vitamin D. The times are taken from a more com­plex analy­sis that depends on time of day and day of the year in areas that experience marked seasons, and the amount of skin exposed to sun. As shown, all skin types can synthesize vitamin D, but the time needed is longer as skin pig­ment­ation increases (higher Fitzpatrick numbers). The times in the southern hemisphere will be influenced by variation in theozone layer thickness.

Table 18b.1 Association of Fitzpatrick skin types with capacity of skin for vitamin D synthesis at 42°N on 21 June at 10:30 hours while exposing one-quarter of body surface area to sunlight. Time needed for vitamin D synthesis in spring or fall equinox or a higher latitude (62.5°N) would be double. Source Webb and Engelsen (2006) Photochemistry and Photobiology 82: 1697–1703.

Fitzpatrick Skin Type	How skin responds to sun exposure	Minutes to make 25µg (1000IU)
I	Always burn never tan	4
II	Burn slightly then tan slightly	6
III	Rarely burn tan moderatelu	7
IV	Never burn, tan moderately e.g. Mediterranean	10
V	Never burn, tan darkly Asian, Indigenous American, Pacific Islander	13
VI	Never burn, tan very darkly; Australian Aborigine, Tamil, West African	21

Sunscreen lotions are used to deliberately block UV rays reaching the skin. Sunscreens are labeled with a sun pro­tect­ion factor (SPF) number which indicates the amount of UV blocked. An SPF blocks at 1/SPF, so that a product having an SPF of 8 would allow only 1/8 (12.5%) of the UV to penetrate the lotion and reach the skin. Theoretically sunscreens should reduce vitamin D syn­thesis. In a nat­ional survey in Canada, however, participants answering “yes” to using sunscreen had higher 25(OH)D levels (2.4 ±1.1nmol/L; P < 0.001) (Brooks et al., 2017). Several reasons may account for this seemingly abherrant finding. Users may apply sunscreens poorly or incompletely (for exam­ple answer “yes” to use but only apply to face). Alternatively, sunscreen users may spend more time outdoors. Other behavioral differ­ences may exist, for exam­ple in the Canadian survey, sunscreen users were more likely to take vitamin D sup­ple­ments than nonusers.

Smoking is assoc­iated with lower serum 25(OH)D con­cent­rations. This may partly explain the repor­ted inc­reased risk of osteoporosis among smokers. The mech­anism is unclear, but the relat­ion­ship does not appear to result exclu­sively from addit­ional con­founding lifestyle factors (Brot et al., 1999). In a large study of adults in Australia, both male and female nonsmokers, includ­ing ex-smokers, had higher mean levels of serum 25(OH)D com­pared to current smokers (Gill et al., 2017).

Obesity, prevalent in many pop­ul­ation groups worldwide, is assoc­iated with a trend towards lower serum 25(OH)D levels (Brooks et al., 2017; Wacker and Holick, 2013). Biologically, this trend can be attrib­uted to vitamin D, whether from cutaneous or diet­ary sources, being deposited in adipose tissue, where it is not bioavail­able (Wacker and Holick, 2013). Some re­searchers have found that obesity in men has less of an effect on reducing 25(OH)D than in women, perhaps because of gender differences in behavioral aspects such as sun avoidance (Rockell et al., 2006). Never­the­less, body weight must be con­sid­ered when evaluating vitamin D status

Disease con­di­tions affect­ing the gastro­intest­inal tract, the liver and kidneys may cause a sec­ond­ary de­fi­ciency of vitamin D (Table 18b.2). Diseases causing fat mal­absorp­tion will reduce the absorp­tion of dietary vitamin D, which could be sig­nif­icant in winter. Liver dis­ease will prevent the con­vers­ion of vitamin D to 25(OH)D, whereas kidney dis­ease prevents the con­vers­ion of 25(OH)D to the active form (i.e. 1,25(OH)₂D, calcitriol) in the endo­crine pathway. Disease states such as in­test­inal mal­absorp­tion and stea­torrhea caused by pan­creatic insufficiency, inflam­matory bowel dis­ease, celiac dis­ease, or mas­sive bowel re­sec­tion have also been associated with lower serum 25(OH)D con­cent­rations. Here, vitamin D deple­tion arises from mal­absorp­tion of diet­ary vitamin D.

Table 18b.2 Secondary causes of vitamin D deficiency. Source Holick (2007). Vitamin D deficiency. The New England Journal of Medicine, 357(3), 266–281.

Causes of Secondary vitamin D Deficiency
Pathology	Diseases
Malabsorption of fat reduces absorption of dietary vitamin D	Cystic fibrosis Celiac disease, Whipple's disease, Crohn's disease, Bypass surgery.
Liver failure prevents production of 25(OH)D	Cirrhosis Hepatitis
Inability to produce 1,25(OH)2D in kidney	Chronic kidney disease
Medications
Drugs reducing Vitamin D absorption	Cholesterol-lowering agents: cholestyramine Weight loss drug orlistat and food additive olestra
Drugs reducing 25(OH)D levels due to increased catabolism	Anticonvulsant medications such as carbamazepine, phenobarbital, and phenytoin, gabapentin. Antiretrovirals agents such as ritonavir and efavirenz, valproic acid (AIDS treatment) Histamine H2 receptor antagonist cimetidine
Drugs Impairing vitamin D metabolism	Oral corticosteroids such as glucocorticoids

Medication use can affect vitamin D status. Any drug which affects liver or kidney cyto­chrome en­zymes will likely affect con­vers­ions of vitamin D meta­bolites. Table 18b.2 provides a list of drugs known to impact vitamin D status. While this list does not cover all possible sec­ond­ary causes of de­fi­ciency, it emphasizes the need to monitor dis­ease states and medi­cation use as possible reasons for vitamin D de­fi­ciency. As is des­cribed below, the require­ment for vitamin D may be ele­vated in persons who have chronic con­di­tions or for whom medi­cation use is required.

Magnesium status may impact 25(OH)D levels and there­fore vitamin D status through the require­ment for two en­zymes of vitamin D meta­bolism: 25(OH)D-1-α-hy­droxy­lase and 25(OH)D-24-hy­droxy­lase. In mag­nes­ium de­fi­ciency, there is a red­uction in the active form 1,25(OH)₂D associated with “Mg-depen­dent vitamin-D-resistant rickets” (Dai et al., 2017). More re­search is needed to deter­mine the intake of mag­nes­ium that affects vitamin D status.

Analytical methods have a marked effect on serum 25(OH)D con­cent­rations. To overcome inter-assay differ­ences, and establish the accuracy and precision of the assay, verified standards should be run with every batch using the Vitamin D Standardization Program. See Section 18b.9.