Christel Lamberg-Allardt

Measurement of 25-hydroxyvitamin D in the clinical laboratory: current procedures, performance characteristics and limitations.

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.

Vitamin D Deficiency and Bone Health in Healthy Adults in Finland: Could This Be a Concern in Other Parts of Europe?

A low vitamin D status could be a concern not only in children and the elderly in Europe, but also in adults. We do not know the effect of mild vitamin D deficiency on bone in this age group. The aim of this study was to detect the prevalence of low serum 25‐hydroxyvitamin D [S‐25(OH)D] and elevated serum intact parathyroid hormone (S‐iPTH) concentrations in healthy young adults in the winter in northern Europe and to characterize the determinants of these variables. In addition, we studied the association between vitamin D status and forearm bone mineral density (BMD) in this population group. Three hundred and twenty‐eight healthy adults (202 women and 126 men, 31–43 years) from southern Finland (60°N) participated in this study conducted in February through March 1998. Fasting overnight blood samples were collected in the morning. Forearm BMD was measured by dual‐energy X‐ray absorptiometry (DXA). The mean daily vitamin D intake met the recommendations in the men (5.6 ± 3.2 μg) and almost met it in the women (4.7 ± 2.5 μg). The mean S‐25(OH)D concentrations did not differ between genders (women, 47 ± 34 nM; men, 45 ± 35 nM; mean ± SD), but the women had significantly higher mean S‐iPTH levels than the men (women, 30 ± 13 ng/liter; men, 24 ± 12 ng/liter; p < 0.001). Low S‐25(OH)D concentrations (<25 nM) were found in 26.2% of the women (53 women) and 28.6% of the men (36 men), respectively. Based on nonlinear regression analysis between S‐25(OH)D and S‐iPTH concentration, the S‐iPTH concentration started to increase with S‐25(OH)D concentrations lower than ∼80 nM in the women and lower than ∼40 nM in the men. Based on this relation between S‐25(OH)D and S‐iPTH concentrations, 86% of the women and 56% of the men had an insufficient vitamin D status. In linear regression analysis, the main positive determinants of S‐25(OH)D were dietary vitamin D intake (p < 0.02), the use of supplements (p < 0.005), alcohol intake (p < 0.05), and age (p < 0.005). Smoking associated negatively with the S‐25(OH)D concentration (p < 0.03). The main determinants of S‐iPTH were S‐25(OH)D (p < 0.01), dietary calcium intake (p < 0.02), and body mass index (BMI; p < 0.01). In addition, female gender was associated with higher S‐iPTH concentration. The mean daily dietary calcium intake was 1037 ± 489 mg and 962 ± 423 mg, in the men and women, respectively. Significantly lower forearm BMD was found in the men (p = 0.01) but not in the women (p = 0.14) with higher S‐iPTH concentrations. Low vitamin D status was prevalent in these young adults in northern Europe in winter, although the vitamin D intake met the recommendation. This probably is not a local problem for northern Europe, because the natural sources of vitamin D are scarce and fortification is not very common in Europe, and with the exception of the southern part of Europe, sunshine is not very abundant in this part of the world. Thus, the results of this study indicate that more attention should be focused on vitamin D status and the sources of vitamin D in these countries.

Vitamin D deficiency in Europe: pandemic?

Background: Vitamin D deficiency has been described as being pandemic, but serum 25-hydroxyvitamin D [25(OH)D] distribution data for the European Union are of very variable quality. The NIH-led international Vitamin D Standardization Program (VDSP) has developed protocols for standardizing existing 25(OH)D values from national health/nutrition surveys. Objective: This study applied VDSP protocols to serum 25(OH)D data from representative childhood/teenage and adult/older adult European populations, representing a sizable geographical footprint, to better quantify the prevalence of vitamin D deficiency in Europe. Design: The VDSP protocols were applied in 14 population studies [reanalysis of subsets of serum 25(OH)D in 11 studies and complete analysis of all samples from 3 studies that had not previously measured it] by using certified liquid chromatography–tandem mass spectrometry on biobanked sera. These data were combined with standardized serum 25(OH)D data from 4 previously standardized studies (for a total n = 55,844). Prevalence estimates of vitamin D deficiency [using various serum 25(OH)D thresholds] were generated on the basis of standardized 25(OH)D data. Results: An overall pooled estimate, irrespective of age group, ethnic mix, and latitude of study populations, showed that 13.0% of the 55,844 European individuals had serum 25(OH)D concentrations <30 nmol/L on average in the year, with 17.7% and 8.3% in those sampled during the extended winter (October–March) and summer (April–November) periods, respectively. According to an alternate suggested definition of vitamin D deficiency (<50 nmol/L), the prevalence was 40.4%. Dark-skinned ethnic subgroups had much higher (3- to 71-fold) prevalence of serum 25(OH)D <30 nmol/L than did white populations. Conclusions: Vitamin D deficiency is evident throughout the European population at prevalence rates that are concerning and that require action from a public health perspective. What direction these strategies take will depend on European policy but should aim to ensure vitamin D intakes that are protective against vitamin D deficiency in the majority of the European population.

Teenage girls and elderly women living in northern Europe have low winter vitamin D status

Objective:To determine the vitamin D status (serum 25-hydroxyvitamin D; S-25OHD) in adolescent girls and elderly community-dwelling women living in four countries of northern Europe and to explain differences in S-25OHD concentrations between and within the countries.Design:A cross-sectional observational study conducted in a standardised way during February–March. S-25OHD was analysed by high-performance liquid chromatography. Vitamin D and calcium intake was calculated using a standardised food composition database.Setting:Denmark, Finland, Ireland, and Poland.Subjects:A total of 199 girls (mean (s.d.) age 12.6 (0.5) y) and 221 women (mean (s.d.) age 71.8 (1.4) y).Results:The median (inter quartiles) concentration of S-25OHD was 29.4 (20.3, 38.3) nmol/l for the girls and 40.7 (28.0, 54.2) nmol/l for the women. S-25OHD below 25 nmol/l was found in 37% of the girls and 17% of the women, and S-25OHD below 50 nmol/l was found in 92% of the girls and 37% of the women. Positive significant determinants for S-25OHD in girls were use of vitamin D supplements, and in women sun habits, dietary vitamin D intake, use of vitamin D and calcium supplements. Body mass index and smoking were negative determinants in women. For women predictors could explain the differences between countries (Pcountry=0.09, R2=0.39), but for girls the difference remained significant even after including predictors (Pcountry=0.03, R2=0.15).Conclusion:Vitamin D status is low in northern Europe during winter. More than one-third of the adolescent girls have vitamin D status below 25 nmol/l and almost all are below 50 nmol/l. Two-thirds of the elderly community-dwelling women have vitamin D status below 50 nmol/l. Use of vitamin D supplements is a significant positive determinant for S-25OHD for both girls and women (P=0.001).Sponsorship:The European Fifth Framework Programme (Contract No. QLK1-CT-2000-00623).

Vitamin D intake is low and hypovitaminosis D common in healthy 9- to 15-year-old finnish girls

Objectives: To study the prevalence of hypovitaminosis D, the effect of vitamin D supplementation on serum 25-hydroxyvitamin D [S-25(OH)D], and the intakes of vitamin D and calcium in Finnish 9− to 15-year-old athletic and nonathletic girls.Design: 1-year follow-up study (February 1997-March 1998) with three months of vitamin D supplementation (10 μg/d) from October to January.Setting: Turku University Central Hospital, Finland.Subjects: 191 female volunteers aged 9–15 y (131 athletes and 60 controls).Methods: Vitamin D and calcium intakes were estimated by a four-day food recording and a semi-quantitative food frequency questionnaire (FFQ). S-25(OH)D was followed by radioimmunoassay (RIA).Results: At baseline the mean S-25(OH)D concentration was 33.9 nmol/l among all girls. In winter severe hypovitaminosis D (S-25(OH)D<20 nmol/l) occurred in 13.4% of the participants and in 67.7% S-25(OH)D was below 37.5 nmol/l. By the next summer the mean S-25(OH)D concentration was 62.9 nmol/l and in 1.6% of the subjects it was below 37.5 nmol/l. The prevalence of severe hypovitaminosis D was not significantly reduced by three months of vitamin D (10 μg/d) supplementation. At baseline, the mean intake of vitamin D was 2.9 μg/d by food recording and 4.3 μg/d by FFQ. The mean calcium intake was 1256 mg/d and 1580 mg/d, respectively. The intakes of vitamin D and calcium remained unchanged during the follow-up period. The athletes consumed more calcium than nonathletic controls, whereas the intake of vitamin D was quite similar among both groups. The vitamin D intake by FFQ correlated with the S-25(OH)D concentration in wintertime (r=0.28, P<0.01).Conclusion: Hypovitaminosis D is fairly common in growing Finnish girls in the wintertime, and three months of vitamin D supplementation with 10 μg/d was insufficient in preventing hypovitaminosis D. The daily dietary vitamin D intake was insufficient (<5 μg/d) in the majority of participants, while the calcium intake was usually sufficient.Sponsorship: Supported by the Yrjö Jahnsson Foundation, The Turku University Foundation, and the Medical Research Foundation of the Turku University Central Hospital.

LONG-TERM CONTROLLED TRIAL WITH DIPHOSPHONATE IN PATIENTS WITH OSTEOLYTIC BONE METASTASES

Thirty-four normocalcaemic women with multiple osteolytic bone metastases from breast cancer were randomly allocated to treatment with disodium dichloromethylene diphosphonate (Cl2MDP) 1600 mg/day orally (17) or placebo (17) for 3-9 months. Fasting urinary hydroxyproline/creatinine and calcium/creatinine ratios declined in the Cl2MDP group but not in the placebo group. Four patients in the placebo group died from hypercalcaemia. New bone metastases were more common in patients on placebo and these patients also required more analgesic drugs than those on Cl2MDP. Cl2MDP seemed to reduce bone pain and bone resorption and prevent the development of hypercalcaemia caused by osteolytic metastases. The formation of new bone metastases and the growth of old ones seemed to be retarded by Cl2MDP.

A Positive Dose–Response Effect of Vitamin D Supplementation on Site-Specific Bone Mineral Augmentation in Adolescent Girls: A Double-Blinded Randomized Placebo-Controlled 1-Year Intervention†

The effect of vitamin D supplementation on bone mineral augmentation in 212 adolescent girls with adequate calcium intake was studied in a randomized placebo‐controlled setting. Bone mineral augmentation determined by DXA increased with supplementation both in the femur and the lumbar vertebrae in a dose‐responsive manner. Supplementation decreased the urinary excretion of resorption markers, but had no impact on formation markers.

High phosphorus intakes acutely and negatively affect Ca and bone metabolism in a dose-dependent manner in healthy young females

Ca and P are both essential nutrients for bone and are known to affect one of the most important regulators of bone metabolism, parathyroid hormone (PTH). Too ample a P intake, typical of Western diets, could be deleterious to bone through the increased PTH secretion. Few controlled dose-response studies are available on the effects of high P intake in man. We studied the short-term effects of four P doses on Ca and bone metabolism in fourteen healthy women, 20-28 years of age, who were randomized to four controlled study days; thus each study subject served as her own control. P supplement doses of 0 (placebo), 250, 750 or 1500 mg were taken, divided into three doses during the study day. The meals served were exactly the same during each study day and provided 495 mg P and 250 mg Ca. The P doses affected the serum PTH (S-PTH) in a dose-dependent manner (P=0.0005). There was a decrease in serum ionized Ca concentration only in the highest P dose (P=0.004). The marker of bone formation, bone-specific alkaline phosphatase, decreased (P=0.05) and the bone resorption marker, N-terminal telopeptide of collagen type I, increased in response to the P doses (P=0.05). This controlled dose-response study showed that P has a dose-dependent effect on S-PTH and increases PTH secretion significantly when Ca intake is low. Acutely high P intake adversely affects bone metabolism by decreasing bone formation and increasing bone resorption, as indicated by the bone metabolism markers.

Bone mineral density and abstention-induced changes in bone and mineral metabolism in noncirrhotic male alcoholics

BACKGROUND AND PURPOSE Abuse of alcohol may derange bone metabolism and cause osteoporosis. Due to confounding factors associated with alcohol abuse, however, the effect of alcohol itself on bone loss remains obscure. The influence of alcohol intake on bone and mineral metabolism is rather well known, but how the metabolism normalizes during withdrawal has rarely been investigated. The aims of the present study were to evaluate the alcohol-induced changes of bone and mineral metabolism and their recovery during abstention, and to reassess any possible link between alcohol abuse and osteoporosis. PATIENTS AND METHODS We studied 27 non-cirrhotic male alcoholics hospitalized for 2 weeks for withdrawal. For comparison, three groups of control subjects were examined. Serum and urinary parameters of bone and mineral metabolism as well as intestinal absorption of calcium were determined at the beginning and end of the treatment period. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry at four axial sites (lumbar spine, femoral neck, Wards triangle, trochanter). RESULTS On admission, bone formation in the alcoholics was reduced as reflected by decreased serum levels of osteocalcin (-28%; p < 0.05) and procollagen I carboxyterminal propeptide (-17%; p < 0.05). Both parameters normalized within 2 weeks of abstention (p < 0.0001 and p < 0.01, respectively). Urinary hydroxyproline, a parameter of bone resorption, was at the control level on admission and increased slightly during abstention (p < 0.05). Serum ionized calcium increased by 3% (p < 0.0001) during withdrawal. Concomitantly, serum free fatty acids (FFA) decreased by 38% (p < 0.001), and there existed an inverse correlation (r = -0.50, p < 0.05) between changes in ionized calcium and FFA. Serum levels of intact parathyroid hormone and vitamin D metabolites were similar in patients and controls throughout the whole observation period. Intestinal absorption of calcium measured by stable strontium was 37% higher in alcoholics than in controls (p < 0.001); it decreased to nearly normal toward the end of the treatment period. Mean axial BMD did not differ between patients and controls at any of the four measurement sites. However, BMD decreased parallel with duration of drinking history in the alcoholics at all axial sites (p < 0.05 to < 0.01, analysis of covariance with age and weight as covariates). CONCLUSIONS Decreased bone formation, which is uncoupled from ongoing bone resorption, recovers completely during 2 weeks of abstention. In the absence of confounding factors, the central BMD is normal in noncirrhotic male alcoholics, although the negative effect of alcohol on BMD is evident when duration of excessive drinking is taken into account.

Vitamin D – a systematic literature review for the 5th edition of the Nordic Nutrition Recommendations

Background The present literature review is part of the NNR5 project with the aim of reviewing and updating the scientific basis of the 4th edition of the Nordic Nutrition Recommendations (NNR) issued in 2004. Objectives The overall aim was to review recent scientific data on the requirements and health effects of vitamin D and to report it to the NNR5 Working Group, who is responsible for updating the current dietary reference values valid in the Nordic countries. Methods The electronic databases MEDLINE and Swemed were searched. We formulated eight questions which were used for the search. The search terms related to vitamin D status and intake and different health outcomes as well as to the effect of different vitamin D sources on vitamin D status. The search was done in two batches, the first covering January 2000–March 2010 and the second March 2009–February 2011. In the first search, we focused only on systematic literature reviews (SLRs) and in the second on SLRs and randomized control trials (RCTs) published after March 2009. Furthermore, we used snowballing for SLRs and IRCTs published between February 2011 and May 2012. The abstracts as well as the selected full-text papers were evaluated in pairs. Results We found 1,706 studies in the two searches of which 28 studies were included in our review. We found 7 more by snowballing, thus 35 papers were included in total. Of these studies, 31 were SLRs and 4 were RCTs. The SLRs were generally of good or fair quality, whereas that of the included studies varied from good to poor. The heterogeneity of the studies included in the SLRs was large which made it difficult to interpret the results and provide single summary statements. One factor increasing the heterogeneity is the large variation in the assays used for assessing 25-hydroxyvitamin D concentration [25(OH)D], the marker of vitamin D status. The SLRs we have reviewed conclude that the evidence for a protective effect of vitamin D is only conclusive concerning bone health, total mortality and the risk of falling. Moreover, the effect was often only seen in persons with low basal 25(OH)D concentrations. In addition, most intervention studies leading to these conclusions report that intervention with vitamin D combined with calcium and not vitamin D alone gives these benefits. It was difficult to establish an optimal 25(OH)D concentration or vitamin D intake based on the SLRs, but there are evidence that a concentration of ≥50 nmol/l could be optimal. The dose–response studies relating vitamin D intake (fortification and supplementation) to S-25(OH)D suggested that an intake of 1–2.5 µg/day will increase the serum concentration by 1–2 nmol/l but this is dependent on the basal concentration with a response being greater when the basal concentration is low. Conclusion Data show that a S-25(OH)D concentration of 50 nmol/l would reflect a sufficient vitamin D status. Results from this review support that the recommendation in NNR 2004 needs to be re-evaluated and increased for all age groups beyond 2 years of age. We refer to the total intake from food as well as supplements, given minimal sun exposure. Limited sunshine, however, does not reflect the situation for the majority of the Nordic population in the summertime. It should also be emphasized that there are large differences in results depending on assay methods and laboratories measuring 25(OH)D, adding to the uncertainty of determining an appropriate target concentration. Moreover, the dose–response of vitamin D on serum 25(OH)D-concentrations is not well established and is dependent on the basal concentrations, sunshine exposure and dietary intake. We advise that these uncertainties should be taken into account when setting the final Nordic recommendations.