Inappropriate serum levels of retinol, alpha-tocopherol, 25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults: simultaneous assessment by HPLC

Clin Biochem. 2008 Jun;41(9):676-80. doi: 10.1016/j.clinbiochem.2008.02.003. Epub 2008 Feb 15.


Objective: Simultaneous assessment of the status of lipid-soluble vitamins; retinol, alpha-tocopherol, 25 hydroxyvitamin D(3) and 24,25 dihydroxyvitamin D(3) in serum of blood donors, paradigm of a healthy population.

Patients and methods: Serum samples were supplied by the Regional Blood Donors Center in Cordoba from 215 healthy Spanish individuals (166 males and 99 females). Target analytes were determined using liquid-liquid extraction and separation-detection by HPLC.

Results: The method was validated using standard reference material (SRM 968c, NIST). Standard errors were 1.4%, 2.1% and 1.8% for 25OHD(3), vitamin A and vitamin E, respectively. The ranges thus assessed were as follows: 17.1+/-8.0 nmol/L, for 24,25(OH)(2)D(3), 40.3+/-34.6 nmol/L for 25OHD(3), 2.57+/-0.7 micromol/L for retinol and 22.13+/-8.30 micromol/L for alpha-tocopherol. Females showed lower serum levels of retinol (p<0.01), alpha-tocopherol (p<0.01) and 25OHD(3) (p=0.028). A total of 10.4% subjects showed vitamin E deficiency, 85.4% had normal levels and 4.2% had high levels of vitamin E. 65.6% of the target subjects showed normal levels of retinol, and 1.6% had moderate or severe vitamin A deficiency. High levels of vitamin A were found in 32.8% of the subjects. Fourteen percent of the healthy subjects showed severe vitamin D deficiency (serum levels of 25OHD(3) <25 nmol/L), 50.8% had vitamin D(3) insufficiency (25OHD(3) from 25 to 50 nmol/L), 17.6% of the subjects had suboptimal 25OHD(3) serum levels (25OHD(3) from 50 to 75 nmol/L), only 16.8% had an adequate status of 25OHD(3) and 0.8% had high levels of vitamin D (25OHD(3)>200 nmol/L). Among subjects with vitamin D below 50 nmol/L, 49.38% had high levels of retinol (over 2.4 mumol/L). This association is considered a risk factor for osteoporosis and fracture.

Conclusions: The reported data of high prevalence of lipid-soluble vitamin values outside the physiological range have important repercussions on public health. These data also uphold the need for simultaneous measurement of fat-soluble vitamins as a valuable tool in clinical practice as well as in epidemiological studies for awareness and correction.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 24,25-Dihydroxyvitamin D 3 / blood*
  • Adolescent
  • Adult
  • Aged
  • Calcifediol / blood*
  • Chromatography, High Pressure Liquid / methods
  • Female
  • Humans
  • Male
  • Middle Aged
  • Spain
  • Vitamin A / blood*
  • Vitamin A Deficiency / blood
  • Vitamin A Deficiency / diagnosis
  • Vitamin E Deficiency / blood
  • Vitamin E Deficiency / diagnosis
  • alpha-Tocopherol / blood*


  • Vitamin A
  • 24,25-Dihydroxyvitamin D 3
  • alpha-Tocopherol
  • Calcifediol