González-Molero

MD Pérez Valero

Md,Hernández

Olveira G Md

MD Gutiérrez Repiso

PhD Rubio Martin

MD F Soriguer

Inmaculada González-Molero
email: inmagonzalezmolero@hotmail.com

Vitamin D deficiency in Spain: a population-based cohort study

Keywords:

Background: Vitamin D deficiency is common worldwide.No homogenous reference values have yet been established and no studies of values have been done in Spain involving a large number of participants. Objective: To study the population concentrations of vitamin D in a representative sample of the Spanish population. Subjects/Methods: The study involved two cohorts from Spain,the Asturias Study and the Pizarra Study,which are two prospective,population-based studies involving 2260 participants.In 1262 subjects (age: 20-83 years) we studied 25-hydroxyvitamin D,intact parathyroid hormone (iPTH),calcium,phosphorus and creatinine. Results: The median population values of 25-hydroxyvitamin D and iPTH were 22.46 ng/mL and 42.29 pg/mL,respectively.The values of 25-hydroxyvitamin D were significantly higher in summer and correlated with age (beta=-0.05±0.01,p<0.0001),creatinine (beta=6.42±1.17,p<0.0001),and iPTH (-0.07±0.01,p<0.0001),but not with calcium,phosphorus or sex.The increase in iPTH with age was seen whatever the values of 25-hydroxyvitamin D,and was greater in the older persons.The concentration of iPTH rose continuously with effect from 25hydroxyvitamin D values below ≈30 ng/mL.Values above ≈35 ng/mL were associated with a significantly lower concentration of iPTH. Conclusions: One third (33.9%) of the Spanish population may be at risk for Vitamin D deficiency.25-hydroxyvitamin D values above 30 ng/mL can safely discard "hyper PTH".The increase in iPTH concentration is greater in older persons for similar values of 25-hydroxyvitamin D.

INTRODUCTION

Previtamin D is synthesized in the skin after sun exposure and can also be ingested with foods having high concentrations of vitamin D.Previtamin D is metabolized in the liver to 25-hydroxyvitamin D and converted in the kidney to the active form 1,25-dihydroxyvitamin D [START_REF] Dusso | Vitamin D[END_REF]Holick,2007).

Vitamin D deficiency can cause a decrease in bone mineral density,secondary hyperparathyroidism and osteomalacia,and it has also recently been associated with numerous other disorders,such as cancer,cancer,diabetes,hypertension,and autoimmune diseases (Holick,2010).

Vitamin D deficiency is common,though its prevalence varies depending on the characteristics of the study population (Adami et al,2009;[START_REF] Annweiler | Is there an association between serum 25-hydroxyvitamin D concentration and muscle strength among older women? Results from baseline assessment of the EPIDOS study[END_REF]Bouuaert et al.,2008;Carnevalle et al.,2001;Hintzpeter et al.,2008;Hintzpeter et al.,2008;Hypponen et al.,2007;[START_REF] Isaia | Prevalence of hypovitaminosis D in elderly women in Italy: clinical consequences and risk factors[END_REF]Lips,2010;[START_REF] Zittermann | An estimate of the survival benefit of improving vitamin D status in the adult German population[END_REF] Despite numerous studies,the reference values of 25-hydroxyvitamin D have not yet been definitively established,and various different cut-off levels have been proposed for the criterion for vitamin D deficiency.A recent consensus document [START_REF] Henry | 14th Vitamin D Workshop consensus on vitamin D nutritional guidelines[END_REF] concluded that the minimum desirable concentration of 25hydroxyvitamin D should be 20-25 ng/mL.

The nutritional deficiency of vitamin D is generally studied by measuring the reserves of vitamin D according to the concentration of 25-hydroxyvitamin D (Holick,2010),either alone or else combined with the measurement of parathyroid hormone (iPTH).

Even though Spain is a country with many hours of sunshine,most studies in Spain show a high prevalence of vitamin D deficiency.However,many of these studies have included persons aged 65 years or over,frequently institutionalized persons (thus,not representative of the general population and with different rates of sun exposure,activity and diet),or else involved only a few persons,with the largest study in the last ten years in the general population [START_REF] Mata-Granados | Inappropriate serum levels of retinol,α-tocopherol,25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults:Simultaneous assessment by HPLC[END_REF] including just 215 persons.Thus,no representative studies involving a large sample of healthy persons have been carried out in Spain.The mean levels of 25-hydroxyvitamin D in middle aged people range from 14-25.9 ng/mL,with 26% to 85.1% of persons having values below 20 ng/ml [START_REF] Calatayud | Prevalencia de concentraciones deficientes e insuficientes de vitamina D en una población joven y sana[END_REF]González Solanellas et al.,2008;[START_REF] Mata-Granados | Inappropriate serum levels of retinol,α-tocopherol,25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults:Simultaneous assessment by HPLC[END_REF].In persons aged 60 years or more,the mean 25-hydroxyvitamin D concentration is even lower: 6.95-17 ng/ml,with 46.4% to 85% having values below 20 ng/ml (Gomez-Alonso et al.,2003;[START_REF] Martín | Niveles de vitamina D en población mayor de 65 años[END_REF][START_REF] Pérez-Llamas | Seemingly paradoxical seasonal influences on vitamin D status in nursing-home elderly people from a Mediterranean area[END_REF][START_REF] Sangrador | The contribution of diet and sun exposure to the nutritional status of vitamin D in elderly Spanish women: the five countries study (OPTIFORD Project)[END_REF][START_REF] Vaquero | Mineral and vitamin status in elderly persons from Northwest Spain consuming an Atlantic variant of the Mediterranean diet[END_REF].

There is a well defined negative correlation between iPTH and 25hydroxyvitamin D [START_REF] Chapuy | Prevalence of vitamin D insufficiency in an adult normal population[END_REF]Cristensen et al.2010;[START_REF] Ho-Pham | Vitamin D status and parathyroid hormone in a urban population in Vietnam[END_REF]Khosla et al.,1997;Leboff et al.,1999;Malaban et al.,1998;[START_REF] Thomas | Hypovitaminosis D in medical inpatients[END_REF]Vieth et al,2003;von Muhlen.,2005),but the features of this relation and the influence of age have not been fully defined.iPTH seems to be higher in older people than in young people [START_REF] Arabi | Age but not gender modulates the relationship between PTH and vitamin D[END_REF][START_REF] Christensen | Seasonal and age-related differences in serum 25-hydroxyvitamin D,1,25-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway[END_REF],but it has been suggested that the relationship between Vitamin D and iPTH is different in older persons compared to young persons,such that for the same levels of 25-hydroxyvitamin D older persons could have higher iPTH [START_REF] Arabi | Age but not gender modulates the relationship between PTH and vitamin D[END_REF][START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF] The aims of this study were,firstly,to examine the values of 25-hydroxyvitamin D in a representative sample of the Spanish population and,secondly,to examine the influence of age in the relation between 25-hydroxyvitamin D and iPTH.

SUBJECTS AND METHODS

Study sample

The study was carried out in two population-based cohorts,one in the north and the other in the south of Spain,both studied at the same time and using similar methods.The study populations and the design of these two surveys have been described previously [START_REF] Soriguer | Prevalence of diabetes mellitus type LADA in South Spain[END_REF][START_REF] Soriguer | Incidence of type 2 diabetes in southern Spain (Pizarra Study)[END_REF]Valdes et al.,2007).The Asturias Study is a prospective,population-based survey of diabetes and cardiovascular risk factors.The baseline examination was carried out during 1998-1999,when 1034 individuals were randomly selected to determine the prevalence of diabetes and prediabetes in the region of Asturias (northern Spain).In 2004-2005,these same subjects were invited for a follow-up examination,700 of whom accepted (Valdes et al.,2007).The Pizarra study is a population-based prospective study undertaken in a population from Andalusia,southern Spain.The first phase of the study (1996)(1997)(1998) included 1226 individuals,selected randomly from the municipal register of Pizarra,Malaga,Spain.Of the original cohort,784 persons were reassessed in 2002[START_REF] Deluca | Overview of general physiologic features and functions of vitamin D[END_REF][START_REF] Soriguer | Prevalence of diabetes mellitus type LADA in South Spain[END_REF][START_REF] Soriguer | Incidence of type 2 diabetes in southern Spain (Pizarra Study)[END_REF].The final sample size was 1484 persons:700 from Asturias,784 from Pizarra).

Persons were excluded from the study if they were institutionalized,had chronic disorders that could affect phosphocalcium metabolism,or if their creatinine was greater than 1.9 mg/dL,calcium >10.9 mg/dL or phosphorus <2.5 mg/dL.Thus,the total number of persons finally studied was 1262.

All the participants completed a clinical survey and underwent an anthropometric study,as well as providing a venous blood sample that was centrifuged at the time of study and the plasma separated and frozen at -80ºC until later analysis.In both cohorts measurements were made simultaneously of 25-hydroxyvitamin D,iPTH,calcium,phosphorous and creatinine in the same laboratory.

The 25-hydroxyvitamin D was measured by electrochemoluminiscence(ECLIA immunoassay,Modular Analytics E170®,Roche).The value is given in ng/mL.The analytical sensitivity was 4-100 ng/mL.Analytical coefficient of variation (CV):7.3% iPTH was measured by electrochemoluminescence(ECLIA immunoassay,Modular Analytics E170®,Roche).The value is given in pg/mL.The assay sensitivity was 1.20-5000 pg/mL.Analytical CV:7.02%.Normal values in our laboratory are:15-60 pg/mL.

The creatinine was measured by spectrophotometry (Dimension

Vista®,Siemens).The value is given in mg/dL.Sensitivity was 0.1-20 mg/dL.Analytical CV:2.7%.

The calcium was measured by spectrophotometry (Dimension Vista®,Siemens).The value is given in mg/dL.The sensitivity was 5-15 mg/dL.Analytical CV:3.7%.

The phosphorus was measured by spectrophotometry(Dimension Vista®

System,Siemens).The value is given in mg/dL (conversion factor: mg/dL x 0.323 = mmol/L).The sensitivity was 0.1-9 mg/dL and the analytical CV was 4.8%.

The solar radiation was measured in watts per square meter(W/m 2 ).The data were obtained from the official information provided by the national meteorological stations.To transform the units into Joules per square meter(Joules/m 2 ),the power in W/m 2 should be multiplied by 86,400(seconds in a day).

The project was approved by the ethics committees of Carlos Haya Hospital in

Malaga and the Hospital Central in Asturias.

Statistical analysis

The continuous variables are presented as the mean and standard deviation or percentiles and the classification variables as proportions.Adjustment of the variables to normality was done with the Shapiro test.The statistical difference between the means of the continuous variables was calculated with the ANOVA test for one or more ways,and for the qualitative variables with the Chi 2 test.

The correlation between variables was measured by calculating the linear coefficient of correlation (Pearson's r) and the regression by designing multiple linear regression models.

The most suitable cut-off point to identify the population reference value of 25-hydroxyvitamin D was calculated by designing ROC curves and calculating the positive and negative predictive value(PPV and NPV).

The strength of association between two variables,adjusted for third variables,was measured using the Odds Ratio (OR),calculated from the coefficient of a logistic regression model.In all cases the level of rejection of a null hypothesis was set at α=0.05 for two tails.(In all models sex was coded as male=0 and female=1)

RESULTS

The mean age of the study participants was 50.3±14.4 years(range:20-83 years),with 57% women and 43% men.The median 25-hydroxyvitamin D and iPTH concentrations were 22.46 ng/mL and 42.29 pg/mL,respectively.Table 1 summarizes the population distribution by percentiles of 25-hydroxyvitamin D,creatinine, iPTH,calcium and phosphorus.

As the age increased,the concentration of 25-hydroxyvitamin D fell (p<0.0001) and that of iPTH rose (p<0.0001),after adjusting for sex,plasma creatinine,individual study (Asturias or Pizarra) and the month of blood extraction (Figure 1).In both men and women the plasma creatinine concentration rose significantly with age,after adjusting for the particular study and the study month (p<0.0001) (R 2 =0.43,p<0.001).The concentration of plasma calcium did not change significantly with age(Data not shown).

The values of 25-hydroxyvitamin D were significantly higher in the summer months in both cohorts (P<0.0001,adjusted for age,sex and creatinine),months that coincide with greater sunlight (Figure 2).In summer,the proportion of persons with 25-hydroxyvitamin D levels >30 ng/mL was greater than in spring or winter: 23.3%,12.8% and 9.3%,respectively (p<0.001 after adjusting the logistic model for age,creatinine,sex and the individual study).The proportion of persons with 25-hydroxyvitamin D values <20 ng/mL was higher in winter than in spring or summer: 37.2%,34.4% and 26.5%,respectively (p<0.001 after adjusting the logistic model for age,creatinine,sex and the individual study).

The values of 25-hydroxyvitamin D rose as the plasma creatinine levels increased,after adjusting for age (p<0.001) and iPTH (p<0.0001).

The values of 25-hydroxyvitamin D were significantly lower in the Pizarra Study than in the Asturias Study (22.75±6.23 ng/mL vs.23.75±7.17 ng/L,p=0.01).In parallel,the values of iPTH were significantly higher in Pizarra than in Asturias (47.00±19.22 pg/mL vs.44.67±16.69 pg/mL; p=0.02).

The concentration of 25-hydroxyvitamin D,iPTH and calcium did not differ significantly depending on sex after adjusting the contrast hypothesis for age and the individual study(data not shown).

A multiple linear regression model showed that the concentration of 25-hydroxyvitamin D correlated significantly with age (beta=-0.03±0.01,p<0.0001),plasma creatinine(beta=6.66±1.17,p<0.0001) iPTH(-0.07±0.01,p<0.0001),and body mass index (BMI) (-0.14±0.03,p<0.000) but not with calcium,phosphorus or sex.The full model explained 8.3% of the variance in the values of 25-hydroxyvitamin D (p<0.0001).

In a multiple linear regression model the iPTH correlated significantly with age (beta=0.27±1.15; p<0.0001),sex(beta=4.07±1.15; p<0.0001),25-hydroxyvitamin D (beta=-0.45±0.07,p<0.0001),plasma creatinine(beta=6.08±3.01,p=0.02),phosphorus(-4.90±1.00,p<0.0001),and BMI(0.32±0.09, p<0.001) but not with calcium.The full model explained 14% of the variance in iPTH(p<0.0001).

In a multiple linear regression model the levels of phosphorus correlated significantly with age (beta=-0.002±0.001,p=0.05),sex (beta=0.22±0.03,p<0.0001),calcium (beta=0.18±0.03,p<0.0001),creatinine(beta=0.17±0.08,p=0.06),and iPTH (beta=-0.004±0.001,p<0.0001) but not with 25-hydroxyvitamin D or BMI.

The increase in iPTH with age was seen whatever the values of 25-hydroxyvitamin D,and were greater in older persons (Figure 3).

Table 2 summarizes the sensitivity,specificity,PPV and NPV,considering the iPTH value (>60 pg/mL) as the biological reference criterion of hypovitaminosis D,for different cut-off values of 25-hydroxyvitamin D.The iPTH levels rose continuously with effect from 25-hydroxyvitamin D values lower than ≈30 ng/mL.Values above ≈35 ng/mL were associated with significantly lower levels of iPTH (Figure 4).

The area under the ROC curve (25-hydroxyvitamin D value according to iPTH) was 0.62±0.021(95% CI,0.58-0.66)(p<0.0001) Logistic regression models: The variable best predicting concentrations of 25hydroxyvitamin D below 20 ng/mL was the value of plasma creatinine(Beta:-1.66;OR 0.19;p<0.0001),followed by the value of iPTH.Inclusion in the model as a dependent variable of another different cut-off point for iPTH (≤65 pg/mL) did not change substantially the strength of the prediction.The variables most strongly associated with values of iPTH (<=60 or >60 pg/mL) were plasma phosphorus (Beta:-0.77; OR:0.46; p<0.0001) and 25-hydroxyvitamin D (Beta:0.53; OR:1.70;p=0.001).

DISCUSSION

If we use the reference value of 20 ng/mL,33.9% of the Spanish population are at risk for vitamin D deficiency,with no significant differences between the study in the north and the study in the south (prevalences of 31.3% and 35.0% respectively).Several studies have been undertaken in Spain on the vitamin D nutritional situation,especially during the 1990s.Most of these studies coincided concerning the presence of vitamin D deficiency,but the prevalence figures for the deficit varied,probably because most were done with opportune or institutionalized samples,and generally involved a low number of persons [START_REF] Calatayud | Prevalencia de concentraciones deficientes e insuficientes de vitamina D en una población joven y sana[END_REF]Gomez-Alonso et al.,2003;González-Solanellas et al.,2008;[START_REF] Mata-Granados | Inappropriate serum levels of retinol,α-tocopherol,25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults:Simultaneous assessment by HPLC[END_REF][START_REF] Pérez-Llamas | Seemingly paradoxical seasonal influences on vitamin D status in nursing-home elderly people from a Mediterranean area[END_REF][START_REF] Vaquero | Mineral and vitamin status in elderly persons from Northwest Spain consuming an Atlantic variant of the Mediterranean diet[END_REF].In other countries in Europe,the percentages of deficiency and insufficiency vary (Lips,2010).Italy has prevalence levels of 17% below 12 ng/ml in winter in the south (Carnevalle et al,2001) and one third of healthy women below 20 ng/ml in the north (Adami et al,2009),which is very similar to our prevalence.Germany has lower levels of vitamin D than in our study: the prevalence of vitamin D insufficiency in the German National Health Interview and Examination Survey was more than 50% in the general population and 60% in children (Hintzpeter et al.,2008;Hitzpeter et al.,2008),with the DEVID (The vitamin in Deutschland) study reporting mean levels of 41±22 nmol/L [START_REF] Zittermann | An estimate of the survival benefit of improving vitamin D status in the adult German population[END_REF].In the British population,the prevalence is greater than in Spain:

Hypponen et al reported a prevalence of hypovitaminosis D <75 nmol/L in 87.1% of participants in winter and in 60.9% in summer (Hypponen et al.,2007).In elderly people,the prevalence of hypovitaminosis D in France,Belgium,Italy and other countries is more than 60% [START_REF] Annweiler | Is there an association between serum 25-hydroxyvitamin D concentration and muscle strength among older women? Results from baseline assessment of the EPIDOS study[END_REF]Bouuaert et al.,2008;[START_REF] Isaia | Prevalence of hypovitaminosis D in elderly women in Italy: clinical consequences and risk factors[END_REF].

In agreement with other studies,we found a negative correlation between levels of 25-hydroxyvitamin D and iPTH (Adami et al.,2010;Arabi et al.2010;Christensen et al,2010;[START_REF] Ho-Pham | Vitamin D status and parathyroid hormone in a urban population in Vietnam[END_REF].On the other hand,the variability in iPTH with seasonal climate changes was lower than that for 25-hydroxyvitamin D,as occurred elsewhere (von Muhlen et al.,2005) and the correlation between iPTH and 25-hydroxyvitamin D was less obvious in spring and summer (data not shown),as in other studies (Maeda et al.,2010),probably related with the different turnover of vitamin D3 and iPTH at a time when the production of vitamin D3 in the skin is greater.In general,most studies assume that iPTH reaches a plateau when 25-hydroxyvitamin D values reach a particular concentration [START_REF] Chapuy | Prevalence of vitamin D insufficiency in an adult normal population[END_REF]Khosla et al.,1997;Leboff et al.,1999;Malaban et al.,1998;[START_REF] Thomas | Hypovitaminosis D in medical inpatients[END_REF]von Muhlen et al.,2005).This observation has led to the use of iPTH levels as a biological marker for the nutritional status of vitamin D (Holick,2007).Others have found no plateau concerning iPTH vs.25-hydroxyvitamin D,suggesting that the choice of an optimum or desirable concentration of 25hydroxyvitamin D,as based solely on iPTH,could be arbitrary (Vieth et al,2003;Ho-Pham et al,2010).Some studies have used linear or exponential models to predict the cut-off point of 25-hydroxyvitamin D values above which the iPTH concentration becomes stable [START_REF] Chapuy | Prevalence of vitamin D insufficiency in an adult normal population[END_REF].In our study the 25-hydroxyvitamin D*iPTH plot was very similar to that reported by others [START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF].However,despite the high level of statistical significance of the various mathematical adjustments,none of the mathematical models used explain more than about 5% of the variance in iPTH (data not shown).In our study,using the cut-off values suggested in the ROC curve,we calculated the predictive values of "hyper iPTH" using various different cut-off points of 25-hydroxyvitamin D.A 25-hydroxyvitamin D cut-off point of ≤30 ng/mL was able to diagnose "hyper iPTH" with a low degree of certainty (18.1%),whereas 25hydroxyvitamin D concentrations above 30 ng/mL were associated with a high degree of certainty (92.8%) at discarding "hyper iPTH".

In our study,as in others,the levels of 25-hydroxyvitamin D fell with age,at the same time as the iPTH increased [START_REF] Arabi | Age but not gender modulates the relationship between PTH and vitamin D[END_REF][START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF] and the phosphorus decreased.Only a few studies have examined the effect of age and kidney function on turnover of 25-hydroxyvitamin D [START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF]Cristensen et al.;[START_REF] Holick | Vitamin D: extraskeletal health[END_REF].In our study,age and plasma creatinine were closely related (r=0.25,p<0.0001) and the levels of 25-hydroxyvitamin D correlated positively with the plasma creatinine,independently of the iPTH levels.In the study by Vieth [START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF],the correlation between age and creatinine was just seen in the older persons,the correlation between 25-hydroxyvitamin D and creatinine only in the younger persons and the change in iPTH correlated with the creatinine only in the older persons,not the younger.Von Mulhen (von Mulhen et al.,2005) on the other hand,found no correlation between creatinine clearance and levels of iPTH or vitamin D.Although most reports have documented a vitamin D deficiency in uremic patients especially due to chronic kidney disease related comorbidities (LaClair et al.,2005 ;[START_REF] Mehrotra | Hypovitaminosis D in chronic kidney disease[END_REF],other have not [START_REF] Craver | Mineral metabolism parameters throughout chronic kidney disease stages 1-5--achievement of K/DOQI target ranges[END_REF].Indeed,25-hydroxyvitamin D levels remained unchanged and levels of 1,25-dihydroxyvitamin D fell as the kidney disease progressed.In another study in patients with normal renal function,levels of vitamin D3 correlated positively with the creatinine [START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF],as they did in our study.We do not have an easy explanation for this issue.It is plausible that a slight impaired renal function may be associated with a lower production of 1-25-hydroxyvitamin D,which induces an increase in iPTH values.The fact that iPTH levels correlated with serum creatinine in our study independently of vitamin D levels,supports this argument.Lastly,we only included patients without chronic clinical disorders but with many hours of sunshine exposure,which may also contribute to maintenance stable 25-hydroxyvitamin D levels,even in presence of slight renal failure.Further studies are needed to clarify this concern.

The fall in 25-hydroxyvitamin D with age seen in this study seems consistent with the findings of others (Arabi et al.2010;[START_REF] Chapuy | Prevalence of vitamin D insufficiency in an adult normal population[END_REF][START_REF] Jacques | Plasma 25-hydroxyvitamin D and its determinants in an elderly population sample[END_REF]Malabanan et al.,1998;[START_REF] Mezquita-Raya | Relation between vitamin D insufficiency,bone density,and bone metabolism in healthy postmenopausal women[END_REF]Von Muhlen.et al.,2005;[START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF],though not all [START_REF] Christensen | Seasonal and age-related differences in serum 25-hydroxyvitamin D,1,25-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway[END_REF][START_REF] Harris | Vitamin D insufficiency and hyperparathyroidism in a low income,multiracial,elderly population[END_REF][START_REF] Tangpricha | Vitamin D insufficiency among free-living healthy young adults[END_REF]).The rise in iPTH levels with age seems to occur whatever the levels of 25hydroxyvitamin D and does not seem to be explained just by the vitamin D3 nutritional status.These results have also been found by others [START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF][START_REF] Arabi | Age but not gender modulates the relationship between PTH and vitamin D[END_REF][START_REF] Holick | Vitamin D: extraskeletal health[END_REF].It seems,therefore,that aging is accompanied by a reduction in the production by the skin of vitamin D3 (MacLaughlin et al.,1985) together with some form of resistance to the effects of 25-hydroxyvitamin D,as has also been seen in older persons for 1,25-hydroxyvitamin D [START_REF] Pattanaungkul | Relationship of intestinal calcium absorption to 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in young versus elderly women: evidence for age related intestinal resistance to 1,25(OH)2D action[END_REF] and proposed by others [START_REF] Vieth | Age-related changes in the 25-hydroxyvitamin D versus parathyroid hormone relationship suggest a different reason why older adults require more vitamin D[END_REF].These reasons,together with the reduction in gut absortion of vitamin D in older persons [START_REF] Bhutto | The clinical significance of gastrointestinal changes with aging[END_REF],may contribute to the changes in levels of 25hydroxyvitamin D and iPTH with age.

If we use a concentration of 25-hydroxyvitamin D as a criterion for nutritional deficiency,e.g.,≤20 ng/mL,those persons studied in summer,spring and winter had a prevalence of vitamin D3 deficiency of 26.9%,34.5% and 37.2%,respectively (p=0.01).However,if we use iPTH ≤60 pg/mL,the prevalence of nutritional deficiency Data are expresed in percentiles.

  serum parathyroid hormone levels and biochemical markers of bone turnover in women: a population-based study.J Clin Endocrinol Metab 82,1522-1527 LaClair RE,Hellman RN,Karp SL,Kraus M,Ofner S,Li Q,et al.(2005).Prevalence of calcidiol deficiency in CKD: a cross-sectional study across latitudes in the United States.Am J Kidney Dis 45:1026-1033. LeBoff MS,Kohlmeier L,Hurwitz S,Franklin J,Wright J,Glowacki J (1999).Occult vitamin D deficiency in postmenopausal US women with acute hip fracture.JAMA 281,1505-1511 Lips P (2010).Worldwide status of vitamin D nutrition.J Steroid Biochem Mol Biol Mar 1.[Epub ahead of print] MacLaughlin J,Holick MF (1985).Aging decreases the capacity of human skin to produce vitamin D3.J Clin Invest 76,1536-1539 Maeda S.,Kunii I,Hayashi L,Lazaretti-Castro M (2010).Increases in summer serum 25hydroxyvitamin D (25OHD) concentrations in elderly subjects in Sao Paulo,Brazil vary with age,gender and ethnicity.BMC Endocrine Disorders 14,10-12 Malabanan A,Veronikis IE,Holick MF (1998).Redefining vitamin D insufficiency. Lancet 351,805-806

Table 2 :Figure 2 :

 22 Figure 1: Concentrations of 25-hydroxyvitamin D (a)

Figure 3 :Figure 4 :

 34 Figure 3: Concentration of iPTH according to age intervals and values of 25-

  Figure 1

Table 1 :

 1 Population distribution of the levels of 25-hydroxyvitamin D,creatinine,iPTH,calcium and phosphorus

	Percentiles

This study was undertaken with finance from the Fondo de Investigacion Sanitaria (PIO51307) and Junta de Andalucía (PIO258/2007,P06-CTS-01684) CIBER de Diabetes y Enfermedades metabólicas is an ISCII project.

Running tittle: Population-based vitamin D values in Spain

was 15.8%,19.9% and 19.8%,respectively,with no significant differences between seasons.Using the measurement of both though,did At this time the aims of an adequate nutritional supplement with vitamin D should be to reach a sufficient concentration of 25-hydroxyvitamin D to achieve levels of iPTH ≤60 pg/mL.However,with the simultaneous measurement of 25-hydroxyvitamin D and iPTH the validation of this objective is far from being satisfactorily achieved.Nonetheless,our results suggest,like others [START_REF] Heaney | Vitamin D: How much do we need,and how much is too much?[END_REF]von Muhlen et al.,2005),that values of 25-hydroxyvitamin D above 30 ng/mL could prevent secondary hyperparathyroidism in most persons.Even so,numerous gaps still remain concerning the best criterion for the diagnosis of vitamin D deficiency.Future prospective studies with large series of populations would therefore appear necessary,using clinical and risk prevention criteria to identify the most suitable criteria to define vitamin D deficiency.

The authors do not have any financial or personal relationships with other people or organisations that could inappropriately influence their work.


Sharon Morris

Mrcophth Mrcsed

MBBCh Richard Barlow

MBBS(Hons), FRACS, FRANZCO Dinesh Selva

Raman Malhotra
email: raman.malhotra@qvh.nhs.uk

Raman Mr

Malhotra

Allergic Contact Dermatitis: a case series and review for the Ophthalmologist

Keywords: Eyelids, immunology, inflammation, diagnostic tests/investigations Word count: 2218

Eyelid dermatitis is most commonly caused by an allergenic response, potentially from exposure at another site, rather than from local toxicity. Yet allergic contact dermatitis (ACD) is a diagnosis often missed by ophthalmologists. We review the literature and detail our experience relating to the causes, clinical features and management of this condition.

Fourteen patients over a 2-year period that were referred to the oculoplastic service for a further opinion, were reviewed in a retrospective, non-comparative study. All patients underwent patchtesting for diagnosis. Eight out of the fourteen patients had delays of greater than 6 months from symptoms to diagnosis. In six of these, this was greater than 1 year. Similar delays are reported in the literature. 79% of our cases were referred by ophthalmologists. Although two of our patients were biopsied, this did not help in making the diagnosis. 13 patients had disease restricted to the eyelids though only five of these had direct contact of the allergen with the eyelids. Two patients were also sensitized to topical steroid creams prescribed for their treatment. All patients improved after removal of the allergen.

Further clinical features and management options from the literature are reviewed and discussed.

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Competing Interest: None to declare.

Introduction

Causes of an itchy, red eyelid include eczema and psoriasis, seborrhoeic dermatitis, meibomitis/blepharitis and rosacea, dermatomyositis, infections, infestations and malignancy.

Allergic contact dermatitis (ACD) is the commonest cause of eyelid dermatitis. [1][START_REF] Valsecchi | Eyelid dermatitis: an evaluation of 150 patients[END_REF][START_REF] Nethercott | A review of 79 cases of eyelid dermatitis[END_REF][START_REF] Katz | Facial dermatitis: patch test results and final diagnoses[END_REF][START_REF] Guin | Eyelid dermatitis: a report of 215 patients[END_REF] Whilst many patients never get to the stage of being referred to an ophthalmologist, up to 13% of all patients with ACD have been reported to present with eyelid involvement. 1,[START_REF] Valsecchi | Eyelid dermatitis: an evaluation of 150 patients[END_REF][START_REF] Nethercott | A review of 79 cases of eyelid dermatitis[END_REF] This may be the only affected site and may result in delayed diagnosis. [START_REF] Katz | Facial dermatitis: patch test results and final diagnoses[END_REF][START_REF] Koo | Solving the mystery of the itchy eyelid[END_REF] Eyelid inflammation tends to be attributed by ophthalmologists more typically, and potentially incorrectly, to local causes, such as topical medications and eye-drops.

Eczema may result from exogenous/endogenous factors. Contact dermatitis is due to exogenous factors and can be irritant or allergic. Irritant contact dermatitis, e.g. toxic reactions to eyedrops, is caused by direct damage and penetration of the skin and represents only approximately 15% of patients with persistent eyelid features. [START_REF] Valsecchi | Eyelid dermatitis: an evaluation of 150 patients[END_REF][START_REF] Nethercott | A review of 79 cases of eyelid dermatitis[END_REF] On the other hand, allergic contact dermatitis is a delayed type IV hypersensitivity reaction to a specific allergen and accounts for the majority. 2,3,6 ,7 ,8 Atopic eczema, which is endogenous, represents only 11-39%. [START_REF] Valsecchi | Eyelid dermatitis: an evaluation of 150 patients[END_REF][START_REF] Nethercott | A review of 79 cases of eyelid dermatitis[END_REF][START_REF] Guin | Eyelid dermatitis: experience in 203 cases[END_REF] Whilst this is well described in the dermatological literature, we believe that ophthalmologists are less aware of the periorbital manifestations and management of this condition. This descriptive study of 14 patients referred to our oculoplastic units for a second/third opinion highlights this delay to diagnosis incurred prior to referral. In addition, we review the current literature and detail the important features of ACD.

Materials and Methods

A retrospective non-comparative case series was performed of patients referred between September 2006 and September 2008 to two specialist oculoplastic centres. 14 consecutive patients with patchtest proven allergens from two independent units were included. The causes, clinical features, management and outcome are described in relation to these patients.

An up-to-date appraised review of the literature was performed, including research from the Ovid Medline and Embase databases. International papers were incorporated. Review articles, large case series and randomised controlled trials were included after appraisal by two separate authors. The main outcome measure was evidence-based literature with clinical relevance.

Results

We studied 14 patients (10 female, age range 5 -72 years). Eleven patients were referred by an ophthalmologist for a second opinion and the remainder by their general practitioners. Clinical details of each patient are included in Table 1. All patients presented with bilateral upper and lower eyelid involvement, with one exception (Case 10) that had unilateral disease secondary to an eyebrow ring.

Most complained of sore, itchy eyelids with swelling of the skin. Some showed lichenification and in two patients (Cases 2 and 6) post-inflammatory hyperpigmentation was the most striking feature. See Figures 1(a-f) and 2(a&b).

The duration of symptoms varied from 2 weeks to 4.5 years (median 1 year, mean 13 months). Eight patients had had symptoms for 6 months or more, of whom five gave a history longer than a year.

Many had tried topical emollients and steroids but it was only upon withdrawal of the allergen that their disease improved. All of our patients were referred to a dermatologist for patch testing.

Two important features of our patients are the range and type of allergens involved. These included: quarternium-15, a constituent of shampoo; nail polish resin; nickel; fragrance-mix; balsam of Peru; benzaklonium chloride 0.1% in lubricant eyedrops; house dust mite; tixocortol pivalate, the steroid component found in hydrocortisone, Eumovate, Betnovate and Dermovate creams; and an excipient in Eumovate cream.

Discussion

Eyelid dermatitis is not uncommon; one author [START_REF] Guin | Eyelid dermatitis: experience in 203 cases[END_REF] reporting involvement in as many as 10% of all general dermatology out-patients. The differential diagnosis includes contact and atopic eczema, seborrhoeic dermatitis, blepharitis, rosacea, psoriasis, dermatomyositis, impetigo and cutaneous T-cell lymphoma.

Acute ACD may present with erythema and macules, papules and/or vesicles. However, blisters are rare on the eyelids. Lichenification, scaling and fissuring are features of more chronic disease. The incidence of ACD as a cause of eyelid dermatitis varies from 29-77% of patients reported, 1,[START_REF] Valsecchi | Eyelid dermatitis: an evaluation of 150 patients[END_REF][START_REF] Nethercott | A review of 79 cases of eyelid dermatitis[END_REF][START_REF] Guin | Eyelid dermatitis: a report of 215 patients[END_REF][START_REF] Ockenfels | Contact allergy in patients with periorbital eczema: an analysis of allergens. Data recorded by the Information Network of the Departments of Dermatology[END_REF][START_REF] Shah | Facial dermatitis and eyelid dermatitis: a comparison of patch test results and final diagnoses[END_REF][START_REF] Ayala | Eyelid dermatitis: an evaluation of 447 patients[END_REF] and has been found to be the most likely cause if all 4 eyelids are involved. [START_REF] Ayala | Eyelid dermatitis: an evaluation of 447 patients[END_REF] . It is more common in middle-aged patients with less pigmented skin. Amin, et al 11 reported 85.4% of their patients with ACD as caucasian in origin with the greatest prevalence in the 41-70 yr old age-range. Females are most frequently affected (61.8% -90% of patients) because of use of cosmetics. 1,[START_REF] Valsecchi | Eyelid dermatitis: an evaluation of 150 patients[END_REF][START_REF] Guin | Eyelid dermatitis: a report of 215 patients[END_REF][START_REF] Ockenfels | Contact allergy in patients with periorbital eczema: an analysis of allergens. Data recorded by the Information Network of the Departments of Dermatology[END_REF][START_REF] Shah | Facial dermatitis and eyelid dermatitis: a comparison of patch test results and final diagnoses[END_REF][START_REF] Ayala | Eyelid dermatitis: an evaluation of 447 patients[END_REF] Immune Process Two stages are necessary in the development of ACD -an initial immune-mediated sensitization to the allergen and then elicitation of the inflammatory response. Sensitization involves penetration of an allergen through the skin and binding to Langerhans antigen-presenting cells. These cells migrate to the lymph nodes and sensitize naïve T lymphocytes, which then relocate themselves back in the skin but throughout the skin. The inflammatory response is elicited by re-exposure to the allergen.

Most environmental allergens are haptens -simple <500d electrophilic molecules that must link to proteins to form a complete antigen before they can sensitize. [START_REF] Landsteiner | Studies on the sensitization of animals with simple chemical compounds, IX: skin sensitization induced by injection of conjugates[END_REF] There are more than 2800 known environmental allergens [START_REF] Degroot | Patch testing: test concentrations and vehicles for 2800 allergens[END_REF] but not all are haptens. If the hapten complexes with a non-immunogenic carrier, then tolerance is induced, rather than sensitization. [START_REF] Katz | Carrier function in anti-hapten antibody responses. IV. Experimental conditions for the induction of hapten-specific tolerance or for the stimulation of antihapten anamnestic responses by "nonimmunogenic" hapten-polypeptide conjugates[END_REF] The carriers for contact allergens are HLA-DR or class II antigens on the surface of Langerhan's cells. [START_REF] Nalefski | Nature of the ligand recognized by a hapten-and carrier-specific, MHCrestricted T cell receptor[END_REF] Because ACD is immune-mediated, compromised immunity is associated with decreased reactivity or anergy. The ageing process modulates ACD, possibly due to a decrease in density of antigen presenting cells and production of proinflammatory cytokines. [START_REF] Belsito | Reversal by lymphokines of the age-related hyporesponsiveness to contact sensitization and reduced Ia expression of Langerhans cells[END_REF] In addition, children and infants can be affected by ACD. It is unclear when immunocompentence is achieved but patch testing has been performed in infants younger than 2 years of age. [START_REF] Motelese | Patch testing in infants[END_REF] More typically, ACD is seen in older children.

Investigation

Skin biopsies are unlikely to distinguish between ACD and other forms of eczema but may help to exclude impetigo and lymphoma. Irritant and allergic contact dermatitis both show spongiosis and a lymphocyte infiltrate. Acute irritant contact eczema usually shows more ballooning degeneration and necrotic keratocytes, whereas ACD shows more spongiosis of the epidermis. [START_REF] Pacor | Tacrolimus ointment in nickel sulphate-induced steroid-resistant allergic contact dermatitis[END_REF] Patch testing is the key investigation used to identify allergens. Frequent contact allergens in eyelid ACD are shown in Table 2. Eyelids are particularly susceptible to ACD because the skin is thinner (thickness of 0.55mm) than on the rest of the face (2.0mm thick). This allows easier penetration of the allergen than at other sites and eyelid dermatitis may therefore be the only manifestation. 11 In addition eyelids may manifest a reaction without direct contact of the allergen at this site.

A thorough history should be taken to identify possible allergens. Details of cosmetics, hobbies and occupation may be relevant. The eyes, eyelids, face and hands (including nails) should be carefully examined.

Patch testing involves application of allergens under Finn Chambers® to the patient's back. Reactions are read at varying intervals. Standard batteries of patch tests, eg. European standard series, TRUE test and North American CD Group (NACDG) series, [START_REF] Marks | North American Contact Dermatitis Group patch test results for the detection of delayed-type hypersensitivity to topical allergens[END_REF] do not include every relevant eyelid allergen and the test should be adjusted for each patient. Guin (2002) found that 66 out of a total of 167 patients with ACD would have remained undiagnosed if the TRUE test was used alone. [START_REF] Guin | Eyelid dermatitis: experience in 203 cases[END_REF] Similarly, Katz [START_REF] Katz | Facial dermatitis: patch test results and final diagnoses[END_REF] found that the TRUE test alone would have detected only 37% of ACD allergens and NACDG only 42% of allergens. The most common relevant allergens are the patient's own personal care products.

Whilst patch testing often provides the answer for the patient, interpretation should be performed by an experienced clinician. Untrained interpretation exposes the patient to incorrect over and undertesting, deceptive results and potentially unwanted sensitization. Interpretation involves being able to separate irritant and allergic reactions, determining the relevance of the antigen and the optimum reading time and appreciation of cross-reactions and co-reactivity.

False positives can occur if the allergen causes an irritant rather than allergic response. The test may need to be repeated with the allergen at a lower concentration. In addition, even if a chemical is found to be allergenic it cannot be assumed that it is causative. The relevance of the antigen is important. A provocation test or repeat open application testing (ROAT) may be necessary. This involves the patient applying the commercial product to normal skin several times daily for 1-2 weeks. [START_REF] Epstein | The use test for contact hypersensitivity[END_REF] Most false negative responses can be avoided by performing a second reading of the test sites 48 hours after the first one. Some studies advocate readings at 4-7 days, especially in elderly patients, to ensure any allergic response is elicited. [START_REF] Przybilla | Evaluation of the immune status in vivo by the 2,4-dinitro-1chlorobenzene contact allergy time (DNCB-CAT)[END_REF][START_REF] Geier | Patch test reactions at D4, D5 and D6[END_REF] Neomycin reactions may take longer -one study showed that half are not evident until 96 hours. [START_REF] Belsito | Reproducibility of patch tests: a US multicenter study[END_REF] In addition, if too low a concentration is used in testing, sensitization may not occur. Sensitization is dependent on dose of chemical per unit area of skin (up to a limit of 0.1cm2). [START_REF] White | The effect of altering area of application and dose per unit area on sensitization by DNCB[END_REF] Concentrations of ophthalmic preparations may need to be tested at a higher level due to difficultly in penetrating the skin on the back. [START_REF] Zug | Dermatological diagnosis and treatment of itchy red eyelids[END_REF] Side effects of patch testing include a severe local reaction or flare reaction at a distant site, an "angry back/excited skin" syndrome where numerous positive reaction occur, pigment changes, scarring and keloids, infections and potentially anaphylaxis. [START_REF] Storrs | Technical and ethical problems associated with patch testing[END_REF] For all of these reasons, patch testing should be performed by an experienced dermatologist. .

Treatment

It is well established that patients may occasionally continue to have symptoms even after avoidance of the allergen. [START_REF] Halbert | Prognosis of occupational chromate dermatitis[END_REF][START_REF] Pryce | Soluble oil dermatitis: a follow-up study[END_REF] Treatment for symptom relief is therefore required in addition to simply identifying responsible allergens.

Treatment should include emollients, treatment of secondary infection if present and down-regulation of the immune response. Topical anti-pruritics should be avoided because of the risk of secondary sensitization. [START_REF] Belsito | The diagnostic evaluation, treatment, and prevention of allergic contact dermatitis in the new millennium[END_REF] Glucocorticosteroids are usually the primary choice for immune modulation and their effective treatment of ACD is well documented. [START_REF] Funk | Horizons in pharmacologic intervention in allergic contact dermatitis[END_REF][START_REF] Hachem | Efficacy of topical corticosteroids in nickel-induced contact allergy[END_REF][START_REF] Queille-Roussel | Objective assessment of topical anti-inflammatory drug activity on experimentally induced nickel contact dermatitis: comparison between visual scoring, colorimetry, laser Doppler velocimetry and transepidermal water loss[END_REF] Inflammation is reduced by suppressing the recruitment of polymorphonuclear leukocytes and reversing capillary permeability. Topical steroids are usually sufficient and treatment should be limited to 2-3 weeks duration. Low potency steroids such as hydrocortisone and desonide are safer on the face, though stronger steroids such as clobetasone proprionate or betamethasone diproprionate are used for moderate-severe disease. [START_REF] Li | Allergic contact dermatitis: pathophysiology applied to future therapy[END_REF] Longstanding application of topical steroids is associated with skin atrophy, telangiectases and acneiform reactions.

If more than 20% of the body surface area is involved, or if there are bullae or extensive facial involvement, then treatment should be considered with systemic steroids.

It is important to be aware that topical steroids may themselves be allergenic, as seen in case 12. One study [START_REF] Gonul | Detection of contact hypersensitivity to corticosteroids in allergic contact dermatitis patients who do not respond to topical corticosteroids[END_REF] of 31 patients with ACD that had worsened or shown no response to topical corticosteroid treatment found that 22% had a positive patch test result to the steroid itself. In other studies, [START_REF] Wilkinson | A prospective study into the value of patch and intradermal tests in identifying topical corticosteroid allergy[END_REF][START_REF] Dooms-Goossens | Results of routine patch testing with corticosteroids series in 2073 patients[END_REF] the steroid has been implicated in 0.2-5%.

Ascomyscins such as tacrolimus (TK506) (Protopic oinment 0.03% or 0.1%) and pimecrolimus (ASM 981) (Elidel cream) have recently been introduced as treatment options and provide a solution for thinskin areas, eg. the face and eyelids. [START_REF] Fleischer | Treatment of atopic dermatitis: role of tacrolimus ointment as a topical noncorticosteroidal therapy[END_REF][START_REF] Sengoku | Possible inhibitory mechanism of FK506 (tacrolimus hydrate) ointment for atopic dermatitis based on animal models[END_REF] They are topical calcineurin inhibitors and cause a reduction in interleukin, leukotriene, histamine and serotonin release, thereby effectively suppressing the immune response. [START_REF] Belsito | The diagnostic evaluation, treatment, and prevention of allergic contact dermatitis in the new millennium[END_REF][START_REF] Nghiem | Tacrolimus and pimecrolimus: from clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis[END_REF] Both agents target the human epidermal Langerhans cell [START_REF] Panhans-Gross | Human epidermal Langerhans' cells are targets for the immunosuppressive macrolide tacrolimus (FK506)[END_REF] and have been shown to inhibit the elicitation phase of ACD in a mouse model. [START_REF] Meingassner | Pimecrolimus inhibits the elicitation phase but does not suppress the sensitization phase in murine contact hypersensitivity, in contrast to tacrolimus and cyclosporine A[END_REF] In addition, a study in humans found that tacrolimus also suppresses the sensitization phase. [START_REF] Lauerma | Inhibition of contact allergy reactions by topical FK506[END_REF] Tacrolimus ointment at concentrations of both 0.03% and 0.1% has been found to be an effective treatment for nickel-induced steroid resistant ACD in adult and paediatric patients. Safety and efficacy of usage has also been reported in children of two years or older. [START_REF] Landsteiner | Studies on the sensitization of animals with simple chemical compounds, IX: skin sensitization induced by injection of conjugates[END_REF] A 0.1% concentration is probably more effective [START_REF] Cheer | Tacrolimus ointment. A review of its therapeutic potential as a topical therapy in atopic dermatitis[END_REF][START_REF] Kang | Long-term safety and efficacy of tacrolimus ointment for the treatment of atopic dermatitis in children[END_REF][START_REF] Reitamo | A multicentre, randomized, double-blind, controlled study of long-term treatment with 0.1% tacrolimus ointment in adults with moderate to severe atopic dermatitis[END_REF] but is more frequently a cause of itching and burning. These rapidly decrease after the first week of treatment.

Although Cyclosporin A is also a successful calcineurin inhibitor, it has limited penetration through the epidermis and limited topical application for this condition. [START_REF] Funk | Horizons in pharmacologic intervention in allergic contact dermatitis[END_REF][START_REF] Surber | Effect of a new topical cyclosporine formulation on human allergic contact dermatitis[END_REF][START_REF] Singh | The effects of dexamethasone, cyclosporine, and vitamin D 3 on the activation of dendritic cells stimulated by haptens[END_REF] Ascomycins have been compared with topical steroids. A small double-blind RCT pilot-study [START_REF] Bhardwaj | A double-blind randomized placebo-controlled pilot study comparing topical immunomodulating agents and corticosteroids for treatment of experimentally induced nickel contact dermatitis[END_REF] examining nickel ACD looked at 4 treatment groups -pimecrolimus 1% cream, tacrolimus 0.1% ointment, clobetasol 0.05% ointment, triamcinolone 0.1% ointment -and two control groups of topical vehicle application. No statistically significant differences were found between any of the groups, although the treatment groups showed a clear trend towards being more effective than control. However, Saripalli et al [START_REF] Saripalli | Tacrolimus ointment 0.1% in the treatment of nickel-induced allergic contact dermatitis[END_REF] induced nickel ACD in patients and found that tacrolimus was significantly more effective than vehicle. Alomar et al 50 corroborated this finding. Similarly, pimecrolimus at 0.2% and 0.6% formulations has successfully treated nickel-induced ACD. [START_REF] Queille-Roussel | SDZ ASM 981 is the first non-steroid that suppresses established nickel contact dermatitis elicited by allergen challenge[END_REF] Other treatments options for more widespread disease away from the eyes, patients that are unresponsive to the above treatments, or for those who cannot avoid the provoking factors include phototherapy -PUVA (oral psoralen photochemotherapy) and shortwave UVB light. In addition, use of Grenz rays [START_REF] Lindelof | The effect of Grenz rays on the expression of allergic contact dermatitis is man[END_REF] and systemic immunosuppressants [START_REF] Sharma | Azathioprine in the treatment of Parthenium dermatitis[END_REF] such as azathioprine and mycophenolate mofatil have been described in ACD.

In our study, all of the patients improved with removal of the allergen with/without a short course of topical immunosuppressants.

In conclusion, eyelid dermatitis may be the only dermatological manifestation of ACD. (See summary table3). Delay in diagnosis commonly hinders appropriate treatment and avoidance of allergens. In our experience, marked delays were due to lack of awareness of the condition by referring ophthalmologists. Improved awareness is essential. In addition, it should be remembered that the corticosteroids used to treat ACD may in fact be causative themselves, and patients who are unresponsive to treatment ought to have corticosteroids included as potential allergens in their patch testing.

Legends to Figures 1a-c: These images correspond to Case 4 in the series who was found allergic to her nail polish. Note the bimedial upper and lower lid erythema and thickening. Examination of her fingernails revealed periungual vesiculation (1c). 1d-f: Images 1d and 1e show another patient from our case series with the typical bimedial upper and lower lid erythema in a 'butterfly' distribution. 1f: Histology specimen from the same patient (H&E stain, x10 magnification). Lichenification, hyperkeratinization, non-specific generalized inflammatory cell infiltate and a thickened cornea stratum are seen. 2a: This patient was referred by an ophthalmologist for upper blepharoplasties, but ACD was found to be causative. Bilateral upper lid erythema is clearly visible in this photo. 2b: This patient demonstrates subtle features of eyelid 'eczematous' dermatitis and medial lower lid ectropion can also be seen. This clinical appearance had prompted repeated prescriptions of steroid cream treatment prior to his presentation to our unit. He was found patch test positive to Eumovate cream.

Table 1 -full details of our case series

 1

	Case Age	Clinical Features	Symptoms to	Patch Testing Positive Results	Management	Outcome
		(years)		diagnosis
	1	46	Sore, itchy upper and lower eyelids with	1.5 years	Quarternium-15, nickel sulphate, caine	Cessation of shampoo, moisturizer and	Dramatic
			erythematous scaling and thickening		mix, formaldehyde, colophony,	make-up products containing allergens.	improvement
			medially in butterfly pattern. No		methylchloroisothiazolinone and	Reducing regime of Betnovate ointment	within 1 month
			benefit with steroid/anti-fungal		methlisothiazolinone, L'Oreal Cleansing	od 2 days, Betnovate RD od for 3 days,
			creams/emollients.		Experience, Lancome Teinte SPF 15.	then 1% hydrocortisone od.
	2	20	Bilateral periocular dermatitis with	4.5 years	Aerosol fragrance-mix	Avoidance of allergen, regular	Periorbital
			cicatricial medial ectropion. Inner			emollients, Betnovate and Eumovate	eczema and
			canthal skin lichenification. No			cream.	entropion
			resolution with steroid ointments or				resolved
			moisturizers to the skin.
	3	74	Itchy eczematous dermatitis of eyelids,	9 months	Caine mix, fragrance mix, nickel and	Avoidance of allergens, Diprobase	Clinically much
			mild eczematous changes to cheeks and		carba mix, E45 itch relief cream,	ointment to wash and moisturize tds,	improved within
			behind ear.		Eumovate (no reaction to	hydrocortisone ointment 0.5 -2.5%	2 months
					Diprobase/hydrocortisone)	depending on redness.
	4	45	Bilateral, itchy, asymmetric	2 months	Nail polish; toluene-sulfonamide	Changed to "hypoallergenic" polyester	Complete
			erythematous eyelids. No other		formaldehyde resin	resin nail polish, 1% hydrocortisone	resolution within
			dermatitis elsewhere on body/face			ointment tds to eyelids.	2 weeks.
			except periungual vesicles on fingers.
	5	72	Mild intermittent erythema and	1 year	Apitol, chloroethylene and ethylene	Stopped mascara wear. No response to	Resolution of
			eczematous eyelid changes.		diamine	Elidel 1% or hydrocortisone 1% use.	symptoms within
						Tacrolimus 0.3% given.	1 month
	6	50	Episodic eyelid rashes, puffiness and	1 year	Nickel, fragrance, balsam of Peru and	Avoidance of allergens.	Complete
			skin darkness.		imidazolidinyl urea preservative		resolution
	7	49	Eyelid puffiness, episodic itchy	3 years	Methylchloroisothiazolinone and	Avoidance of products.	Improved within
			erythematous eyelid skin. Occasional		methlisothiazolinone, from shampoo and		2 months
			neck rash.		her Crème de la Mere foundation
	1

Table - full details of our case series (continued)

 -

	Case Details	Clinical Features	Symptoms to	Patch Testing Positive Results	Management	Outcome
			diagnosis
	8	Bilateral periocular dermatitis, itchy lids	4 months	Fragrance-mix	Ceased aftershave lotion, hydrocortisone	Resolved within
		with some lichenification.			1% ointment tds to eyelids.	4 weeks
	9	Periocular dermatitis and bilateral	2 weeks	Benzalkonium chloride 0.1%aq.	Topical lubricants stopped, predsol 0.5%	2 week
		conjunctival inflammation since starting			qds both eyes and topical hydrocortisone	resolution
		ocular lubricants.			1% tds to eyelid skin.
	10	Left-sided eyelid and eyebrow	4 weeks	Nickel	Removal of piercing, 0.1%	Complete
		dermatitis, following insertion of			betamethasone valerate ointment bd.	resolution
		eyebrow ring.
	11	Bilateral lichenified itchy eyelids.	6 months	House dust mite	Measures to decrease exposure	Partial resolution
					(mattress/pillow protectors, cleaning of	over 2 months
					floors, open windows daytime, washing
					stuffed toys etc.). Topical
					hydrocortisone 1% tds to skin.
	12	Bilateral upper and lower eyelid	14 months	Tixocortol pivalate (found in	Avoidance of allergens.	Full resolution
		erythema and oedema. Failed treatment		hydrocortisone, Eumovate, Betnovate		within 2 months
		with hydrocortisone 1% ointment.		and Dermovate ointments), quaternium-
				15
	13	Bilateral upper lid erythema -referred	6 months	Parabens and lanolin	Avoidance of face creams. Reducing	Complete
		for consideration of blepharoplasties.			regime of hydrocortisone 1% ointment.	resolution
	14	Sore, itchy, erythematous upper and	1 year	Nickel sulphate	Avoidance of her eyelash curlers. No	Complete
		lower eyelids.			topical creams necessary.	resolution

Table 2 -

 2 Common contact allergens particularly relevant to eyelid ACD

	Allergen

Table 3 - Summary Table 8 9

 3Summary8 Recommended

4-step Approach to Management of Suspected ACD

	1 History	Ask about known allergens, types of cosmetics,
		occupational and leisure pursuits. Remember
		allergens may not necessarily be those in direct
		contact with the eye.
	2 Examination	Eyelids and nails -may not have localized nailbed
		changes. Check for artificial nails.
	3 Refer for	Standard patch testing batteries should be
	patch testing	supplemented with patient's own cosmetics or
		particular allergens from history. Allow 48-96
		hours prior to result reading.
	4 Treatment	Cessation of allergen contact.
		Symptom relief -emollients, topical antipruritics,
		oral antihistamines.
		A 2-3 week course of topical steroids or
		tacrolimus/pimecrolimus use.

Acknowledgements

Thanks are given to Eduardo Calonje and Bill Barrett for their assistance in histopathological examination.


PhD Kevin Whelan
email: kevin.whelan@kcl.ac.uk

MSc Sasha Dunn

MSc Artika Datta

MSc Sophia Kallis

Emma Law

Clio Myers

Emma Law Msc

Clio E Myers Bsc

Validation of a food frequency questionnaire to measure intakes of inulin and oligofructose

Keywords: Food frequency questionnaire, fructo-oligosaccharides, validity, reliability, inulin, oligofructose

INTRODUCTION

Inulin-type fructans are non-digestible carbohydrates consisting of D-fructose monomers linked by β2→1 bonds to form linear oligomers and short polymers.

Although terminology varies, inulin can describe a variety of molecules covering the full range of polymerisation, from 2-60, whereas oligofructose can refer to shorter chain lengths ranging from 2-10 [START_REF] Roberfroid | Prebiotic effects: metabolic and health benefits[END_REF].

Inulin and oligofructose are associated with numerous health benefits via their prebiotic effects, defined as 'the selective stimulation of growth and/or activities of one or a limited number of micro-organisms in the gut microbiota that confer health benefits to the host' [START_REF] Roberfroid | Prebiotic effects: metabolic and health benefits[END_REF]. Supplements of inulin or oligofructose increase gastrointestinal (GI) concentrations of bifidobacteria [START_REF] Meyer | The bifidogenic effect of inulin and oligofructose and its consequences for gut health[END_REF], and they may positively influence appetite [START_REF] Cani | Gut microbiota fermentation of prebiotics increases satietogenic and incretin gut peptide production with consequences for appetite sensation and glucose response after a meal[END_REF], immune function [START_REF] Lomax | Prebiotics, immune function, infection and inflammation: a review of the evidence[END_REF] and mineral absorption [START_REF] Scholz-Ahrens | Prebiotics, probiotics, and synbiotics affect mineral absorption, bone mineral content, and bone structure[END_REF]. Despite these effects, high intakes can induce GI symptoms, including mild flatulence and borborygmi [START_REF] Bruhwyler | Digestive tolerance of inulintype fructans: a double-blind, placebo-controlled, cross-over, dose-ranging, randomized study in healthy volunteers[END_REF].

Given the health effects of inulin and oligofructose, measuring their intake from diet is of considerable importance, but is impeded by a lack of food composition data.

Accurate data is available only for the major food commodity sources [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF][START_REF] Campbell | Selected fructo-oligosaccharide (1kestose, nystose, and fructofuranosylnystose) composition of foods and feeds[END_REF], although recent data has reported the inulin content of selected Australian fruits and vegetables [START_REF] Muir | Fructan and free fructose content of common Australian vegetables and fruit[END_REF][START_REF] Muir | Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)[END_REF].

However, there remains little data for composite foods.

Consequently, only two studies have ever quantified dietary inulin and oligofructose intakes, both were from the 1990's and both used food composition data from Van [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF]. The first reported the average daily inulin intake to be between 1-4 g/d in the US and 3.2-11.3 g/d across Europe [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF]. Subsequently, [START_REF] Moshfegh | Presence of inulin and oligofructose in the diets of Americans[END_REF] used data from two 24-h recalls, and also included intakes from composite foods by using standard recipes from the Food Commodity Intake Database (FCID). Mean daily intakes of inulin and oligofructose were 2.6 g/d and 2.5 g/d, respectively. Intakes in the United Kingdom have not been measured.

The problems associated with accurate dietary assessment are well documented [START_REF] Bingham | Comparison of dietary assessment methods in nutritional epidemiology: weighed records v. 24 h recalls, food-frequency questionnaires and estimated-diet records[END_REF]. Although weighed food diaries may be relatively accurate, they require high subject motivation and lengthy data handling. Food frequency questionnaires (FFQs) can provide an accurate, rapid and inexpensive method of dietary assessment and are frequently used in epidemiological studies. FFQs have been developed that measure intake of all nutrients [START_REF] Mckeown | Use of biological markers to validate self-reported dietary intake in a random sample 16 of the European Prospective Investigation into Cancer, United Kingdom, Norfolk cohort[END_REF][START_REF] Lassale | Estimating food intakes in Australia: validation of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) food frequency questionnaire against weighed dietary intakes[END_REF], specific nutrients (e.g. folate, [START_REF] Pufulete | Validation of a short food frequency questionnaire to assess folate intake[END_REF], food commodities (e.g. fruit, vegetables, [START_REF] Petkeviciene | Validity and reproducibility of the NORBAGREEN food frequency questionnaire[END_REF] or dietary components (e.g. quercetin, [START_REF] Ranka | Development of a food frequency questionnaire for the assessment of quercetin and naringenin intake[END_REF]. However, in a number of settings, short-term dietary assessment is required. Therefore, some FFQs have been validated to measure intakes over the short-term (e.g. 7 days, [START_REF] Eck | Precision and estimated accuracy of two short-term food frequency questionnaires compared with recalls and records[END_REF][START_REF] Eck | Measuring short-term dietary intake: development and testing of a 1-week food frequency questionnaire[END_REF] and can therefore be used for dietary assessment during clinical trials [START_REF] Xinying | Can a food frequency questionnaire be used to capture dietary intake data in a 4 week clinical intervention trial?[END_REF]).

An FFQ that measured short-term intakes of inulin and oligofructose would allow between-group comparisons in dietary surveys, as well as the measurement of background dietary intakes in inulin or oligofructose supplementation trials. The aim of this study was therefore to accurately measure inulin and oligofructose intakes in a group of healthy subjects and to develop and validate an FFQ to measure shortterm intake.

METHODS

Development of the FFQ

Inclusion of items in the FFQ was determined following extensive review of the literature. The standard food composition data available at the time of FFQ development was used [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF], and was consistent with that used in the two previous dietary surveys [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF][START_REF] Moshfegh | Presence of inulin and oligofructose in the diets of Americans[END_REF].

Subsequently published food composition data has not identified additional major dietary sources of inulin and oligofructose [START_REF] Muir | Fructan and free fructose content of common Australian vegetables and fruit[END_REF][START_REF] Muir | Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)[END_REF].

Based upon this food composition data, eight fruit and vegetables were included in the FFQ (asparagus; banana; chicory root; garlic; globe artichoke; Jerusalem artichoke; leeks; and onions). Chicory coffee was also included because where consumed it would likely have a large impact on intakes. The cereals wheat, barley and rye contain small amounts of inulin and oligofructose but are widely consumed.

Therefore, wheat-containing composite foods were eligible for inclusion in the FFQ where the UK National Diet and Nutrition Survey indicated their mean intake to be greater than 100 g/week or where they were consumed by more than 50% of the sample population [START_REF] Henderson | National Diet and Nutrition Survey: adults aged 19-64[END_REF]. This resulted in the inclusion of breads, breakfast cereals, pizza, pasta, noodles, bulgar wheat/cous-cous, cakes/muffins, pastry products, puddings and biscuits. Composite food sources of rye (e.g. rye breads) and barley (e.g. breakfast cereals, beer) were also included in the FFQ. Some of the composite food items were split to allow for ethnic and portion size variations (e.g. 'bread, sliced' and 'other breads'). This resulted in an FFQ that included 23 food items (8 fruits/vegetables; 15 composite foods).

To maximise correct identification of each food item, all composite foods included suggestions of products (for example, 'Pastry products e.g. meat pie, cheese pasty, apple pie, Danish pastry'). A photograph flashcard was available for asparagus, globe artichoke, Jerusalem artichoke and chicory root for subjects who did not recognise the item's name.

The FFQ requested a typical portion size for each of the 23 food items (small, medium, large), which were described in household measures. The weight of each was taken from standard food portion size tables for the UK [START_REF] Crawley | Food Portion Sizes[END_REF]. Where data was not available, the foods were purchased and the research team weighed an agreed portion size.

The FFQ then requested the frequency of consumption of each item during the previous 7-d. This period was chosen because the prebiotic effects and GI sideeffects of inulin and oligofructose result from current/recent intake, rather than historic intake, and because this would facilitate the measurement of background intakes in supplementation trials, which may be only 7-d in length, and is therefore Measuring intakes over the previous 7-d is therefore likely to be of greater clinical and research utility.

Measurement dietary intake

Healthy subjects were recruited through a circular email sent to all students and staff at King's College London and through the snowballing technique. The inclusion criteria were adults aged 18-50 years who identified themselves as healthy. Broad inclusion criteria were used in order to recruit a representative population in which to validate the FFQ [START_REF] Cade | Development, validation and utilisation of food frequency questionnaires-a review[END_REF]. Exclusion criteria were any acute or chronic diseases (e.g. diabetes, GI disorders), antibiotic use within the last 3 months, regular medication use (except the contraceptive pill), prebiotic or probiotic use within the previous month or following a special/restrictive diet. This study was approved by the King's College London Research Ethics Committee and written informed consent was obtained from all subjects. Subjects' height and weight were measured and their BMI calculated. Subjects were asked to complete a 7-d semi-weighed food diary (FD), and were provided with a FD, electronic weighing scales accurate to 1 g (Soehnle, Switzerland). They were instructed to record the weight and a detailed description of all food/drinks consumed. Where food weighing was not possible (e.g. restaurants) subjects were asked to estimate consumption using the validated Photographic Atlas of Food Portion Sizes [START_REF] Nelson | Food portion sizes: a photographic atlas[END_REF][START_REF] Nelson | Food photography II: use of food photographs for estimating portion size and the nutrient content of meals[END_REF]. Subjects were asked to consume their normal diet over the 7-d period. Detailed verbal and written instructions were provided by nutritionists.

Data from the 7-d FD were entered into a computerised dietary analysis package (MicroDiet, v1.2, UK) to calculate nutrient intakes. Basal metabolic rate was estimated using the modified Schofield equations (DH, 1991). Finally, energy intake:basal metabolic rate (EI:BMR) was calculated [START_REF] Black | Critical evaluation of energy intake using the Goldberg cut-off for energy intake:basal metabolic rate. A practical guide to its calculation, use and limitations[END_REF].

Measuring inulin and oligofructose intakes

Following completion of the 7-d FD, subjects were asked to complete the FFQ on day 8 (FFQ1) and again on day 9 (FFQ2). As the FFQ requested information on food intake over the previous seven days, subjects were asked to complete them at the end of the 7-d FD period in order that intake was being measured during the same period by both methods. Dietary analysis software does not currently contain information regarding inulin and oligofructose content of foods. Therefore, intakes were calculated in a two-stage process consistent with that used in a previous comprehensive dietary survey [START_REF] Moshfegh | Presence of inulin and oligofructose in the diets of Americans[END_REF]. Firstly, the quantity of all food and drinks from the 7-d FD (e.g. banana, lasagne) was converted into the quantity of inulin and oligofructosecontaining commodities (e.g. banana, wheat, onions, garlic) using the 2004 revised FCID (DoA/EPA, US). These were then converted into consumption using the midpoint value for inulin and oligofructose content of each food commodity [START_REF] Moshfegh | Presence of inulin and oligofructose in the diets of Americans[END_REF]. The average daily intake of inulin and oligofructose over the 7-d period was then calculated for each subject.

For the FFQ, the calculation of inulin and oligofructose consumption was automated through the development of a programmed spreadsheet (MS Excel, Microsoft, US). This converted portion size and frequency for the 23 food items into the quantity of inulin/oligofructose-food commodities consumed, and then into average daily inulin and oligofructose intakes, using the same data as above.

Statistical analysis

Mean (SD) inulin and oligofructose intakes were calculated. Concurrent validity was assessed by comparing intakes from FFQ1 with that of the 7-d FD using paired t-tests and Pearson's correlation coefficients (PCC). Bland-Altman plots were also generated and the mean difference (FFQ1-FD) and limits of agreements (2 SD of the mean difference) were interpreted [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]. The n (%) of subjects classified into the same or adjacent tertiles by the FD and FFQ1 was calculated, and misclassification was reported as the n (%) categorised into opposite tertiles by the two methods.

Intra-observer reliability was assessed by comparing intakes measured using FFQ1 and FFQ2, using paired t-tests and Pearson's correlation coefficients. The agreement between categorical variables (portion size, frequency) on FFQ1 and FFQ2 were calculated using percentage agreement and the kappa statistic. Each kappa statistic was compared with recognised standards of agreement as follows: 'no agreement' (κ<0); 'slight' (κ=0.21-0.40); 'fair' (κ=0.21-0.40); 'moderate' (κ=0.41-0.60); 'substantial' (κ=0.61-0.80); and 'almost perfect' (κ=0.81-1.00) [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF].

All statistical analysis was conducted on SPSS (v17 SPSS Inc, Chicago, US) and a P value of ≤0.05 was considered statistically significant.

RESULTS

In total, 66 healthy subjects (17 males, 49 females) were recruited, with a mean age of 31 yrs 2 months (SD 7 y, 11 m) and the sample included eight current smokers and nine vegetarians (Table 1). Fifteen had a BMI≥25kg/m², of which eleven were female.

Males had significantly higher energy and fat intakes than females. The group EI:BMR was 1.47 (0.34).

Mean inulin and oligofructose intakes were 4.0 (1.3) g/d and 3.8 (1.2) g/d respectively. Wheat was the largest contributor to both inulin (66.2% ± 16.5) and oligofructose (69.2% ± 15.5) intakes, with onion (17.3% ± 10.6 and 18.2% ± 10.8), and garlic (5.4% ± 7.3 and 2.5% ± 4.2) also making significant contributions.

Validity

There were no significant differences in inulin intake (4.0 ± 1.3 vs 4.0 ± 1.4 g/d, P= 0.646) or oligofructose intake (3.8 ± 1.2 vs 3.8 ± 1.3 g/d, P=0.864) when measured using the 7-d FD or the FFQ1, respectively (Table 2). There was a positive correlation between intakes from the 7-d FD and the FFQ1 for both inulin (PCC=0.406, P=0.001) and oligofructose (PCC=0.403, P=0.001).

Intakes were compared between the 7-d FD and the FFQ1 using Bland-Altman distributions. For inulin, the difference between the two methods was -0.09 (1.5) g/d and the limits of agreement ranged from -3.1 to +2.9 g/d (Figure 1a). For oligofructose, the mean difference was -0.03 (1.4) g/d and the limits of agreement ranged from -2.9 to 2.8 g/d (Figure 1b). There was no pattern in the placement of outlying values, suggesting no systematic bias in measurement using the FFQ.

The FFQ categorised the majority of subjects into the same (50%) or adjacent (39%) tertile of inulin intake as the 7-d FD. It also categorised the majority of subjects into the same (44%) or adjacent (45%) tertile of oligofructose intake. Therefore only 11% of subjects were misclassified into opposite tertiles.

Intra-rater reliability

There were no significant differences in inulin intake (4.0 ± 1.4 vs 3.9 ± 1.7 g/d, P= 0.505) or oligofructose intake (3.8 ± 1.3 vs 3.7 ± 1.5 g/d, P=0.419) between FFQ1 and FFQ2, respectively (Table 2). In addition, there were strong positive correlations between intakes from FFQ1 and FFQ2 for both inulin (PCC=0.759, P<0.001) and oligofructose (PCC=0.748, P<0.001).

Comparing FFQ1 and FFQ2, the agreement between subjects' 'usual portion size' ranged from 50% (Jerusalem artichoke) to 100% (beer, globe artichoke, chicory root, chicory coffee). Although it could not be calculated for all food items, the resulting κ values ranged from 0.39 to 1.00. Therefore, agreement was classified as 'almost perfect' (eight items), 'substantial' (nine items), 'moderate' (two items) or 'fair' (one item) [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF] Table 3).

Comparing FFQ1 and FFQ2, the agreement between subjects' 'frequency of consumption' ranged from 62% (breads) to 100% (globe artichoke, chicory root, chicory coffee). It was rarely possible to calculate κ values, but where calculated, they ranged from 0.55 to 0.86, with agreement being classified as 'almost perfect' (one item), 'substantial' (three items) or 'moderate' (two items) (Table 3).

DISCUSSION

Inulin and oligofructose are prebiotic carbohydrates naturally present in the diet, and yet there are no convenient, yet accurate, methods of measuring dietary intakes.

The reason for measuring inulin and oligofructose intakes using a 7-d semi-weighed FD was to provide a standard against which to compare the data from the FFQ. The sample is largely female, young and potentially educated and health conscious, and therefore is unrepresentative of the entire UK population. Despite these limitations, the actual inulin and oligofructose intakes are interesting, intensively recorded, and the first reported in a UK population. Intakes of inulin (4.0 g/d) were generally higher than in the US (2.6 g/d, [START_REF] Moshfegh | Presence of inulin and oligofructose in the diets of Americans[END_REF]1-4 g/d, Van Loo et al., 1995), but were at the lower end of the range of intakes across Europe (3.2-11.3 g/d, Van Loo et al., 1995).

These differences may relate to real differences in dietary intake between the populations reported. For example, artichokes are concentrated sources of inulin but contributed only 1.2% of inulin intake, compared with 6.3-7.1% in the European sample [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF], potentially reflecting higher intakes of such items among non-UK Europeans. Although it contains relatively small quantities, wheat was the largest contributor to inulin and oligofructose intake, supporting previous findings [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF][START_REF] Moshfegh | Presence of inulin and oligofructose in the diets of Americans[END_REF], likely due to its frequent consumption in the Western diet. Differences in the findings between studies may also relate to differences in dietary assessment used, with the most comprehensive being the 7-d FD used here.

However, the high recording burden meant that our sample population was inevitably much smaller, may have resulted in self-selection of healthier subjects and may have resulted in subjects altering their food intake [START_REF] Vuckovic | A qualitative study of participants' experiences with dietary assessment[END_REF]. These issues support the development of an FFQ that measures inulin and oligofructose intake.

It was not possible to use biomarkers to measure concurrent validity in the current study due to the absence of such a marker of inulin and oligofructose intake. It was therefore necessary to validate the FFQ against a standard dietary assessment method. The FFQ demonstrated good validity, with population intakes almost identical to the FD and correlations between the two methods similar to those reported in other validation studies [START_REF] Cade | Food-frequency questionnaires: a review of their design, validation and utilisation[END_REF]. The Bland-Altman analysis demonstrated very low mean differences between the FD and FFQ for measuring inulin (-0.09 g/d) and oligofructose (-0.03 g/d) intakes within a population, but with relatively large limits of agreement, its assessment of intakes in individuals may be subject to error. However, when ranking into tertiles, only 11% of subjects were misclassified, which is similar to values obtained in other validation studies [START_REF] Petkeviciene | Validity and reproducibility of the NORBAGREEN food frequency questionnaire[END_REF][START_REF] Ambrosini | The reliability of a food frequency questionnaire for use among adolescents[END_REF].

The reliability of the FFQ would also appear to be good, with no differences in inulin and oligofructose intakes when measured on different days (FFQ1, FFQ2) and strong correlations between the two. Agreement between FFQ1 and FFQ2 for both 'portion size' and 'frequency' indicates that the FFQ is highly reproducible for measuring inulin and oligofructose intake.

In order that all dietary assessment related to a similar time period, FFQ1 and FFQ2 were completed one day apart, and followed completion of a 7-d FD. This could result in elevated measured validity due to improved recall of intake following the 7d FD and elevated reliability due to memory of the information recorded on FFQ1 [START_REF] Cade | Food-frequency questionnaires: a review of their design, validation and utilisation[END_REF]. However, these aspects of design were to ensure dietary assessment over similar periods, and are a necessary limitation of other validation studies of FFQs that measure short-term intake [START_REF] Eck | Precision and estimated accuracy of two short-term food frequency questionnaires compared with recalls and records[END_REF][START_REF] Eck | Measuring short-term dietary intake: development and testing of a 1-week food frequency questionnaire[END_REF].

A number of issues must be considered when undertaking dietary assessment using any FFQ, and these also created methodological dilemmas in the current study. Some subjects omitted information (i.e. portion size, frequency), and such omissions were coded as zero, as it has been shown that this is most frequently the actual intake [START_REF] Michels | Self-administered semi-quantitative food frequency questionnaires: patterns, predictors, and interpretation of omitted items[END_REF]. Interestingly, in the current study, one subject recorded consumption of 42 medium slices of bread on FFQ1, but failed to complete the portion size on FFQ2, resulting in a large loss of information. Therefore, omitted information should be minimised wherever possible. Given the natural variation in inulin and oligofructose content of foods [START_REF] Van Loo | On the presence of inulin and oligofructose as natural ingredients in the western diet[END_REF], another limitation is the use of the midpoint value, and is a problem in all studies that rely on such food composition data. Furthermore, the FFQ measures the intake of inulin and oligofructose that is naturally occurring in foods. Due to their potential health benefits, inulin and oligofructose are now being added as functional ingredients in food products such as yoghurts and breakfast cereals. The availability, formulation and use of such food products varies widely between different countries [START_REF] Franck | Technological functionality of inulin and oligofructose[END_REF]. Therefore, those wishing to use the FFQ should first identify the availability of such fortified food products and record their intake alongside the FFQ in order to ensure accurate assessment of inulin and oligofructose intakes.

Although 7-d weighed FD may be the most accurate dietary assessment method, they may change actual dietary intake and are impractical in many clinical trial settings. The 24-h dietary recall requires trained investigators and may not collect data that sufficiently reflects actual dietary intake. Although FFQs were not original designed to do so, they may facilitate short-term dietary assessment, as long as they are stringently validated. Therefore, despite the limitations discussed, we conclude that the FFQ developed here is a rapid, valid and reliable method for measuring inulin and oligofructose intakes. It will facilitate short-term dietary surveys and will be particularly important for the measurement of dietary inulin and oligofructose intakes in supplementation trials.

Figure 1a Bland-Altman plot of inulin intakes measured using a 7-d semi-weighed FD and FFQ1 (n=66). The mean difference (FFQ1-FD, solid line) is -0.09 g/d and the Bland-Altman limits of agreement (2 SD of the mean difference, dashed lines) are -3.1 to 2.9 g/d.

Figure 1b

Bland-Altman plot of oligofructose intakes measured using a 7-d semiweighed FD and FFQ1 (n=66). The mean difference (FFQ1-FD, solid line) is -0.03 g/d and the Bland-Altman limits of agreement (2 SD of the mean difference, dashed lines) are -2.9 to 2.8 g/d.

TABLE 1

 1 Characteristics of 66 healthy subjects

	Mean (SD)	Males	Females	P value	Total
		(n=17)	(n=49)	(t-test)	(n=66)
	Age (y, m)	32y, 10m (7, 11)	30y, 7m (7, 5)	0.305	31y, 2m (7, 6)
	BMI (Kg/m 2 )	24.1 (3.8)	22.9 (2.9)	0.156	23.2 (3.1)
	Nutrient intake
	Energy (kcal/d)	2406 (413)	2019 (458)	0.003	2119 (476)
	Protein (g/d)	95.3 (17.7)	80.2 (46.0)	0.194	84.1 (41.1)
	Fat (g/d)	90.7 (19.0)	76.7 (18.7)	0.010	80.3 (19.6)
	Carbohydrate (g/d)	283.0 (68.9)	248.7 (60.8)	0.057	257.5 (64.2)
	NSP (Englyst, g/d)	10.4 (7.0)	12.9 (7.5)	0.234	12.3 (7.4)
	Nutrient intake was measured using a 7-d semi-weighed FD

TABLE 2

 2 Comparison of mean (SD) inulin and oligofructose intakes measured using the 7-d semi-weighed food diary (7-d FD), FFQ1 and FFQ2.

		Intake measured using different	P value
			methods (g/d)
	n=66	7-d FD	FFQ1	FFQ2	7-d FD vs FFQ1 FFQ1 vs FFQ2
	Inulin	4.0 (1.3)	4.0 (1.4)	3.9 (1.7)	0.646	0.505
	Oligofructose 3.8 (1.2)	3.8 (1.3)	3.7 (1.5)	0.864	0.419

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the US Department of Agriculture and the US Environmental Protection Agency for the use of the Revised FCID. Valuable advice regarding the inulin-type fructans was provided by Prof Marcel Roberfroid (Université Catholique de Louvain, Brussels, Belgium). The authors wish to thank all subjects for participating in this study.

CONFLICT OF INTERESTS

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. There are no conflicts of interest. recorded and therefore the total number of responses (N) was less than 66. 4

However, for 'frequency of consumption' missing values were assumed 'zero' and 5 therefore the total number of responses (N) was always 66. 6 21 -κ values could not be calculated as they require a symmetric 2-way table in which the values of the first variable match the values of the second.