Milk fortified with the current adequate intake for vitamin D (5 microg) increases serum 25-hydroxyvitamin D compared to control milk but is not sufficient to prevent a seasonal decline in young women

Abstract

Low vitamin D status in women of childbearing age may have implications for health. Vitamin D status of New Zealanders (NZ), based on low serum 25-hydroxyvitamin D (25OHD) is suboptimal. Vitamin D status may be improved with supplements and/or fortified foods. Recently an Adequate Intake (AI) for Australia and NZ was set at 5 microg/d vitamin D. We aimed to determine the effect of daily consumption of milk powder fortified with 5 microg vitamin D3 on serum 25OHD concentration over 12 wks. 73 non-pregnant women (18 - 47 y) living in Dunedin, NZ (46 degrees S) were randomised to receive either unfortified (control) or fortified (5 microg vitamin D3) milk for 12 wks from January to April. Mean 25OHD was similar between groups at week 0 (control 74 vs 76 nmol/L) and fell significantly in both groups over the 12 weeks (control 53 nmol/L, fortified 65 nmol/L; p < 0.001). After 12 wks the fortified milk group had a serum 25OHD 19% (95% CI; 7, 32%) higher (10 nmol/L) than the control group after adjusting for baseline levels (p < 0.001). Daily consumption of fortified milk providing the current AI of 5 microg day vitamin D3 for 12 weeks resulted in higher 25OHD concentrations than control milk. This dose was not sufficient to prevent the seasonal decline in 25OHD. This study suggests an AI of 5 microg may be inadequate for New Zealanders to allow for seasonal changes in sunlight exposure, and is unlikely sufficient for other populations with low sunlight exposure.

Full text

Asia Pac J Clin Nutr 2010;19 (2):195-199 195
Original Article
Milk fortified with the current adequate intake for
vitamin D (5μg) increases serum 25-hydroxyvitamin D
compared to control milk but is not sufficient to prevent
a seasonal decline in young women
Tim J Green PhD1,2, C Murray Skeaff PhD1, Jennifer E Rockell PhD1
1Department of Human Nutrition, University of Otago, Dunedin, New Zealand
2Food, Nutrition, and Health, University of British Columbia, Vancouver, BC, Canada
Low vitamin D status in women of childbearing age may have implications for health. Vitamin D status of New
Zealanders (NZ), based on low serum 25-hydroxyvitamin D (25OHD) is suboptimal. Vitamin D status may be
improved with supplements and/or fortified foods. Recently an Adequate Intake (AI) for Australia and NZ was
set at 5 µg/d vitamin D. We aimed to determine the effect of daily consumption of milk powder fortified with 5
µg vitamin D3 on serum 25OHD concentration over 12 wks. 73 non-pregnant women (18 - 47 y) living in Dune-
din, NZ (46°S) were randomised to receive either unfortified (control) or fortified (5 µg vitamin D3) milk for 12
wks from January to April. Mean 25OHD was similar between groups at week 0 (control 74 vs 76 nmol/L) and
fell significantly in both groups over the 12 weeks (control 53 nmol/L, fortified 65 nmol/L; p < 0.001). After 12
wks the fortified milk group had a serum 25OHD 19% (95% CI; 7, 32%) higher (10 nmol/L) than the control
group after adjusting for baseline levels (p < 0.001). Daily consumption of fortified milk providing the current
AI of 5 µg per day vitamin D3 for 12 weeks resulted in higher 25OHD concentrations than control milk. This
dose was not sufficient to prevent the seasonal decline in 25OHD. This study suggests an AI of 5 µg may be in-
adequate for New Zealanders to allow for seasonal changes in sunlight exposure, and is unlikely sufficient for
other populations with low sunlight exposure.
Key Words: 25-hydroxyvitamin D, milk, vitamin D, New Zealand, women
INTRODUCTION
Vitamin D deficiency leads to rickets in children1 and
osteomalacia in adults.2 Lesser forms of vitamin D defi-
ciency, often termed insufficiency, may increase the risk
of hyperparathyroidism, osteoporotic fracture and other
negative health outcomes.3 The few natural food sources
of vitamin D such as fatty fish are not regularly consumed
by the population. Thus in the absence of vitamin D forti-
fication or supplementation the major source of vitamin D
is endogenous skin synthesis by UV light exposure. Any-
thing that influences the amount of light reaching skin,
such as season, latitude, clothing, and darker skin colour
will influence vitamin D status. Recent surveys indicate
high rates of vitamin D insufficiency in many countries
including Australia and New Zealand, based on low circu-
lating concentrations of 25 hydroxyvitamin D (25OHD).4,5
For example, 80% of New Zealand adults in the 1997
National Nutrition Survey had a serum 25OHD less than
75 nmol/L with even higher rates of insufficiency in the
winter months and among Maori and Pacific People.5
Due to inadequate UV light in the winter months and
health concerns about UV light exposure, food fortifica-
tion with vitamin D may be a practical strategy for im-
proving vitamin D status.6 A Vitamin D intake recom-
mendations, termed an Adequate Intake, for Australia and
New Zealand were recently set at 200 IU/d (5 µg/d) for
all people less than 50 y.7 While overseas studies suggest
this level of intake may be too low to maintain circulating
25OHD concentrations at a level adequate for bone
health,3 the effect of 5 ug vitamin D3 on New Zealanders’
circulating 25OHD concentrations has not been deter-
mined.
Here we present results from a double blind random-
ised controlled trial investigating the effect of a fortified
milk supplement containing 5 µg vitamin D3 on serum
25OHD and intact parathyroid hormone (PTH) concentra-
tions in NZ women of childbearing age.
MATERIALS AND METHODS
Subject Recruitment
Seventy-three women volunteers from Dunedin aged 18-
45 years were recruited through advertisements in local
newspapers and signs placed around the university.
Corresponding Author: Dr Tim Green, Food, Nutrition, and
Health, University of British Columbia, 2205 East Mall,
Vancouver, BC, Canada V6T 1Z4.
Tel: +1 604 822 0421; Fax: +1 604 822 0421
Email: tim.green@ubc.ca
Manuscript received 25 August 2009. Initial review completed
25 December 2009. Revision accepted 8 March 2010.

196 Foritifed milk and 25OHD in women
Women were excluded if they consumed a vitamin and/or
mineral supplement in the previous three months, or had
established chronic disease. We also excluded women who
had been pregnant in the previous year, or were planning
a pregnancy. The Human Ethics Committee of the Uni-
versity of Otago approved the study and all women gave
written informed consent to participate.
Intervention
This was a 12-week, double blind, randomised controlled
trial beginning in January. Women were asked to attend
an early morning clinic at the Department of Human Nu-
trition (week 0). The women were weighed, had their
height measured and asked to complete a demographic
and lifestyle questionnaire. Women were randomised to
one of two treatment groups (fortified or control milk).
They were provided with verbal and written instructions
on how to prepare the milk powder and to complete a
diary of their milk consumption. Participants were asked
to return any unused milk powder on their return to clinic
12 weeks after baseline (April, week 12). Compliance
was assessed by dividing the number of servings of milk
consumed by the number of possible servings.
Milks
Fonterra Brands (Auckland, New Zealand) provided the
fortified milk (ANMUM Materna™) and the control milk.
The control milk powder was a mixture of whole milk
and skim milk powders that were blended to match the fat
level of the fortified milk. Participants were instructed to
consume 75 g of milk powder daily as 37.5 g powder in
200 ml water twice daily (morning and evening). This
amount (75 g) of fortified milk powder provided 200 IU
(5 µg) of vitamin D3. The control milk contained a negli-
gible amount of vitamin D. The milks (75 g of powder)
provided daily; 1330 KJ and 9 g of fat, 5 g of which was
saturated fat.
Laboratory Assessment
Blood was drawn from subjects following an overnight
fast at week 0 and week 12. Blood samples were proc-
essed for storage within 4 hours of collection; Blood was
centrifuged (2500×g, 7 min) and the serum was aliquoted
to cryovials. All samples were stored at –70˚C until
analysis, immediately following the intervention. Serum
25OHD was determined using a DiaSorin radioimmuno-
assay (Stillwater, MN). Serum PTH concentrations were
also determined using radioimmunoassay kits (DiaSorin
Stillwater, MN). Two levels of control provided by the
manufacturer were run in each assay. Inter- and intra-
assay coefficient of variations based on repeated analysis
of pooled controls for vitamin D were 11% and 9%, re-
spectively; and for PTH 11% and 7%, respectively. Sam-
ples from individuals were run as pairs to minimise intra-
assay variation.
Data Analyses
Data was log transformed and presented as geometric
means wit 95% confidence intervals. We defined vitamin
D insufficiency as a 25OHD less than 75 nmol/L.8 The
difference in blood measurements over the 12 weeks was
assessed using a paired t-test. The difference in the preva-
lence of insufficiency over the 12 weeks was assessed by
McNemar’s chi-squared test. The difference in measure-
ments between the treatment group and the control group
at week 12 were determined by regression analysis, con-
trolling for baseline values. Logistic regression with ad-
justment for baseline prevalence was used to determine
the odds ratio for the difference in the prevalence of
25OHD less than 75 nmol/L between the fortified and
placebo milk groups at week 12. A p-value of <0.05 was
used to indicate significance.
RESULTS
Of the 73 women randomised to treatment, seven with-
drew from the study, five in the fortified milk group and
two in the control milk group. Women withdrew for the
following reasons; pregnancy (n=1), anaemia (n=1), gas-
tro-intestinal disturbances (n=2), dislike of the milk (n=3).
The majority of participants were non-smoking, young
adult women of European ethnicity (Table 1).
At baseline the mean (95% CI) 25-hydroxyvitamin D
concentration did not differ between control [74 (65, 85)
nmol/L], and fortified [76 (66, 87) nmol/L] milk groups
(Table 2). By Week 12 the 25OHD concentrations had
declined to 53 (46, 62) nmol/L in the control milk group
and 65 (57, 73) nmol/L in the fortified milk group (p <
0.001). There was a 19% (7, 32) (p <0.001) difference in
25OHD concentrations at 12 weeks after adjusting for
baseline values, which translates to a mean difference of
10 (4, 20) nmol/L (p=0.001). At baseline 44% of women
in the control group and 47% in the treatment group had a
25-hydroxyvitamin D concentration less than 75 nmol/L.
By 12 weeks the prevalence of insufficiency significantly
increased to 79% (p <0.001) in the control milk group and
was 53% in the fortified milk group (p=0.727)], with an
adds ratio of 0.18 (p=0.011) for fortified versus control
milks, adjusted for baseline values. Mean (95% CI) PTH
concentrations did not differ between control [2.8 (2.6,
3.1) pmol/L] and fortified milk groups [2.9 (2.7, 3.1)
pmol/L] and did not change over the course of the study.
DISCUSSION
Women consuming fortified milk had 10 nmol/L higher
serum 25OHD concentrations than those consuming the
placebo at 12 weeks, which translates to a mean treatment
effect of ~2 ug/nmol/L 25OHD concentration. O’Donnell
et al recently conducted a systematic review of the effects
Table 1. Characteristics of study participants in each
treatment group.
Placebo milk Fortified milk Baseline
characteristic (n=36) (n=37)
Age (years) 28.8 (26.3, 31.3) 28.0 (25.5, 30.6)
Weight (kg) 64.9 (61.2, 68.7) 67.4 (63.2, 71.2)
BMI (kg/m2) 23.7 (22.4, 25.0) 23.3 (22.9, 25.8)
Smokers, n (%) 1 (8.3) 2 (5.6)
Ethnicity, n (%)
European 33 (91.7) 29 (78.4)
Asian 2 (5.6) 6 (16.2)
Indian 1 (2.8) 2 (5.4)
Compliance to
treatment, % 96 (95, 98) 95 (92, 98)
Values are means (95% CI) unless otherwise stated

TJ Green, CM Skeaff and JE Rockell 197
of fortified foods (primarily dairy products) on 25OHD
concentrations. Of 9 studies, which included a total of
889 subjects, all but one reported a significant beneficial
effect on 25OHD concentrations.6 The individual treat-
ment effects ranged from 14.5 to 34.5 nmol/L with 3.4 to
25 µg vitamin D per day, respectively. Our findings and
those of O’Donnell et al indicate that the effect of vitamin
D fortified foods on 25OHD concentrations is larger per
ug of intake than that previously reported for vitamin D
supplementation trials.6 For example, in a six-month vi-
tamin D3 supplementation trial, Aloia predicted that for
every ug intake of vitamin D3 there would be a 0.7 nmol/L
rise in 25OHD somewhat lower than in our study.9 How-
ever, the lowest dose given was 50 ug/d and lower doses
appear to have a greater effect on 25OHD change per ug
than higher doses. Our findings suggest that vitamin D3
added to milk is more bioavailable than a supplement,
however no direct comparisons have been made to prove
this.
Although 5 µg of vitamin D provided by the fortified
milk resulted in higher 25OHD relative to control it was
not sufficient to arrest the seasonal decline in serum
25OHD concentrations. While the prevalence of insuffi-
ciency did not change significantly in the treatment group,
it would likely have increased had the study continued for
longer than 12 weeks. The decline in serum 25OHD vi-
tamin D concentrations between baseline and 12 weeks in
women consuming the control milk was expected. A sea-
sonal variation in 25OHD is well described in people liv-
ing at higher latitudes. Circulating 25OHD concentrations
are typically at their highest in early Autumn and then fall
until early Spring.10 This study commenced in late Janu-
ary and finished in late April. This allowed us to deter-
mine 25OHD concentrations at their maximum for the
year, and to examine how the current AI of 5 ug/d might
attenuate the seasonal decline in 25OHD. The 25OHD
concentrations at baseline and at 12 weeks in the women
receiving the control milk were similar to women 25 and
44 years of age surveyed in the 1997 National Nutrition
Survey. In that survey mean 25OHD concentrations in
this group were of 87 nmol/L in January and 59 nmol/L in
April.5
Parathyroid hormone concentrations are known to rise
in the presence of low dietary calcium intake and/or low
vitamin D status causes inadequate absorption of calcium
from the diet, resulting in calcium being sourced from the
skeleton to maintain circulating calcium. Seasonal fluc-
tuations in parathyroid hormone concentration have been
reported in New Zealanders between the months of Feb-
ruary and October but there was no change in parathyroid
hormone concentrations by 12 weeks (April) in either
group of this study.11 It may be that a longer time period
including winter months is necessary in order to detect
changes in parathyroid hormone concentrations in New
Zealand women. Further, the 1000 mg additional calcium
provided by the milks may have attenuated a rise in para-
thyroid hormone concentration with decreasing 25OHD.12
Our study had a number of limitations. We included
only women of childbearing age only and our results can-
not necessarily be extrapolated to other adults. Men had
higher 25OHD concentrations than women in the 1997
New Zealand National Nutrition Survey.5 Further,
25OHD concentrations declined with age in New Zealand
women suggesting that older women may require more
vitamin D to maintain serum 25OHD concentrations.5
However, in a study of older people (n=92) living in an
Australian residential care facility, 25OHD concentra-
tions increased by greater than 3 nmol per µg vitamin D
with supplementation.13 Our study was only 12 weeks in
duration and we don’t know whether 25OHD concentra-
tions had reached a plateau in the fortified group. Finally,
there is uncertainty around the most appropriate cut-off
for 25OHD to define vitamin D insufficiency. We chose
75 nmol/L, but cut-offs between 50 to greater than 100
nmol/L have been recommended.14,15
In conclusion, daily consumption of fortified milk pro-
viding the current AI of 5 µg day vitamin D3 for 12 weeks
resulted in higher serum 25OHD concentrations than con-
trol milk. This dose, however, was not sufficient to pre-
vent the seasonal decline in 25OHD. Our findings would
seem to support the view of many experts that the current
AI is insufficient to maintain optimal vitamin D status. If
larger amounts of vitamin D are required to maintain op-
timal vitamin D- and our results suggest they are- consid-
Table 2. Serum 25-hydroxyvitamin D (25OHD), and intact parathyroid hormone (PTH) concentrations during the
trial1
Insufficiency, 25OHD %<75 nmol/L
Baseline Week 12 % Difference2Baseline Week 12 Odds Ratio3
25OHD nmol/L
Placebo milk 74 (65, 85) 53 (46, 62) * 44 (27, 61) 79 (65, 93)**
Fortified milk 76 (66, 87) 65 (57, 73) * 19 (7, 32)†† 47 (29, 65) 53 (35, 71) 0.18 (0.05, 0.68)†
Intact PTH pmol/L
Placebo milk 2.8 (2.6, 3.1) 2.9 (2.7, 31)
Fortified milk 2.9 (2.7, 3.1) 2.9 (2.7, 3.2) -0.1 (-0.9, 0.8)
Values are geometric means (95% CIs).
1Placebo group, baseline n=36, wk 12 n=34; fortified milk group, baseline n=37, wk 12 n=32
2Percent (95%CI) difference in 25OHD and PTH concentration at wk12 determined by regression analysis adjusting for baseline values
3Odds ratio for difference in prevalence of 25OHD % <75 nmol/L (95%CI) determined by logistic regression adjusting for baseline preva-
lence
*Significantly different from baseline, paired t-test; p <0.05
**Significantly different from baseline, McNemar's chi square test for prevalence; p <0.001
†Significantly different from placebo milk; p <0.05
††Significantly different from placebo milk; p <0.001

198 Foritifed milk and 25OHD in women
eration may need to be given to fortifying a greater range
of foods with vitamin D in New Zealand or recommend
that people take a vitamin D containing supplements.
AUTHOR DISCLOSURES
Fonterra Brands (Auckland, New Zealand) funded the study and
provided the milk powders. TJG has consulted for Fonterra
Brands Limited. There were no other conflicts of interest.
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TJ Green, CM Skeaff and JE Rockell 199
Original Article
Milk fortified with the current adequate intake for
vitamin D (5μg) increases serum 25-hydroxyvitamin D
compared to control milk but is not sufficient to prevent
a seasonal decline in young women
Tim J Green PhD1,2, C Murray Skeaff PhD1, Jennifer E Rockell PhD1
1Department of Human Nutrition, University of Otago, Dunedin, New Zealand
2Food, Nutrition, and Health, University of British Columbia, Vancouver, BC, Canada
維生素D (5 μg) 強化的牛奶提高了血清25-羥維生素D水
平但不足以預防年輕女性維生素D水平的季節性下降
維生素D不足會對育齡婦女的健康產生影響。由分析血清25-羥維生素D (25OHD)
得知，紐西蘭人的維生素D營養狀態並不理想。攝取補充劑與/或者食品強化都可
以提高維生素D水平。目前澳洲和紐西蘭的維生素D適宜攝入量(AI)訂為每日5
μg。本研究檢測，每日食用添加5 μg維生素D3的奶粉12周後，對血清25OHD水
平的影響。居住在紐西蘭但尼丁(46°S)的73名未懷孕婦女(18-47歲)，隨機分配食
用未強化奶粉(對照組)或者強化奶粉(5 μg維生素D3)，從1月到4月連續12周。在
第0周，兩組婦女的平均血清25OHD值相似(74對照76 nmol/L)。12周後，平均血
清值均明顯下降(對照組53 nmol/L，強化奶粉組65 nmol/L，p<0.001)。調整血清
25OHD基礎值之後，食用強化奶粉12周的婦女血清25OHD值比對照組高19%
(95% CI；7，32%)，即10 nmol/L (p<0.001)。與對照組相比，連續12周每天食用
添加5 μg維生素D3的牛奶可以提高血清25OHD水平。但該劑量不足以預防
25OHD的季節性下降。該研究顯示，對於紐西蘭人而言，5 μg的適宜攝入量(AI)
也許不足以彌補陽光照射的季節性差異。這個攝入量對於其他陽光照射較少地區
的族群也很可能是不足的。
關鍵字：25-羥維生素D、牛奶、維生素D、紐西蘭、婦女