Development of laboratory tests for assessing vitamin status

Abstract

Aim To develop assays for the determination of functional vitamin status and to determine the functional vitamin status of an elderly population. Specifically the focus has been to develop assays to measure the biological concentrations of N,N dimethylglycine and glycine betaine as possible markers of functional folate status.

Background Functional vitamin deficiencies arise when the tissue concentration of a vitamin derived coenzyme is inadequate this can occur despite normal concentrations of the vitamin in the blood or urine, and leads to lower activity of the vitamin dependent enzyme. The lowered enzyme activity causes changes in the concentrations of metabolites associated with the biochemical pathway catalysed by the enzyme. The amount of vitamin at a tissue level can be determined by measuring these changes. Functional vitamin deficiencies have been associated with many chronic diseases. Further investigations of the relationships between nutrients create a need for appropriate assays.

Methods and results Two new trifluromethanesulfonate reagents (2-phenanthrenacyl and 6-methoxynapthacyl) have been synthesised. These reagents form highly fluorescent derivatives with N,N dimethylglycine, and betaines including glycine betaine and propionylcarnitine. Using 2 phenanthrenacyl triflate as the derivatising reagent the detection limit for glycine betaine is improved from 0.2 μM to 0.04 μM. Optimisation is achieved by changing the solvent, base and water content of the reaction mixture. Polar aprotic solvents are used, with the presence of some water or alcohol tolerated. Suitable bases include the inorganic bases, magnesium hydroxide, silver oxide and lithium phosphate.

The cationic derivatives of N,N-dimethylglycine and glycine betaine in plasma are separated by HPLC on an alumina column within 50 minutes (Between batch CV < 20%). Cation exchange HPLC is carried out using a polar organic solvent containing an aqueous buffer with an organic cation and a hydrophilic anion. Selectivity is affected by the choice of organic solvent and buffer. Increasing the water content and the buffer concentration reduces the retention of the derivatives. Propionylcarnitine can be quantified after separation by HPLC on a non endcapped strong cation exchange column however the use of this assay to detect functional biotin deficiency has not been investigated.

1H NMR can be used to measure N,N-dimethylglycine and glycine betaine in urine. The inter and intra-assay CV’s were < 10% and recoveries were ≥ 97% over a linear range from 50 μM to 1000 μM. Limits of detection using 1H NMR spectroscopy (15 – 25 μM) are higher than HPLC assays, though adequate for the detection of raised concentrations in urine.

Elderly hip fracture patients (aged 65-90) were investigated, as they are known to have poor nutrition compared to health elderly, and would be expected to have associated vitamin deficiencies. A greater percentage of hip fracture patients had insufficient vitamin B12 and folate concentrations compared to age matched healthy elderly controls (Folate 55% and 32% <120 pM, Vitamin B12 7% and 5% <8.5 nM). The results for other analytes are difficult to interpret because of the affects of recent trauma in the hip fracture population. In the control population glycine betaine predicts total homocysteine concentrations (multiple linear regression –0.055 P = 0.099) and is a stronger predictor of folate than total homocysteine. The ratio of the concentrations of N,N dimethylglycine/glycine betaine was not significantly associated with folate status.

Conclusions The concentrations of N,N-dimethylglycine and glycine betaine in plasma can be measured by HPLC, and in urine by 1H NMR. In healthy elderly there is a high prevalence of vitamin (vitamin B12 and folate) deficiency. The ratio of N,N-dimethylglycine/glycine betaine is not an appropriate marker of functional folate status, however the associations between glycine betaine and homocysteine metabolism require further investigation.