M. J. M. Jongen

Histomorphometric profile and vitamin d status in patients with femoral neck fracture

Abstract In order to detect metabolic bone disease vitamin D metabolites and other biochemical parameters of bone metabolism were measured in 125 patients with femoral neck fracture and 74 age-matched control subjects. Transilial bone biopsies from 89 patients, obtained within 1 week of the fracture, were evaluated by histomorphometric techniques. Trabecular bone volume (TBV) was significantly lower in trochanteric fractures in both sexes than in controls, whereas in cervical fractures TBV was not decreased. Other histomorphometric differences between both fracture types were not observed. Cortical porosity was higher in the patients than in autopsy controls. The osteoid surface was extended in the patients, but the osteoid seams were significantly thinner than in controls. The mean width of osteoid seams was not increased in any patient. On these grounds serious osteomalacia is unlikely. With remodeling parameters the biopsies were classified into three subgroups: “high turnover” ( n =19), “uncoupling” between resorption and formation ( n =9), and a “rest” group ( n =61), in which bone turnover was normal or low. In the “high turnover” group mean cortical thickness was decreased significantly compared with the other groups. Mean serum concentrations of calcium (corrected for serum proteins), phosphate, creatinine, alkaline phosphatase, and parathyroid hormone were not different in patients and control subjects. Serum concentrations of the vitamin D metabolites 25(OH)D, 24,25(OH)2D, and 1,25(OH) 2 D were significantly lower in patients than in controls. The histomorphometric picture could not be predicted adequately by means of biochemical parameters. An increased osteoid volume (≥ 5%), and/or surface (≥ 25%), observed mainly in the “high turnover” group, was always associated with a serum 25(OH)D concentration below 30 nmol/l. The “high turnover” may follow vitamin D deficiency. The latter, however, does not cause a clear histological reaction in most cases.

The effect of trauma on serum concentrations of vitamin D metabolites in patients with hip fracture

In a previous study we observed lower serum concentrations of 25(OH)D, 24,25(OH)2D, and 1,25(OH)2D in patients with hip fracture than in aged control subjects. In order to evaluate the effect of trauma on vitamin D metabolite levels, we measured serum concentrations of vitamin D binding protein (DBP) in 118 patients with hip fracture and 71 aged control subjects. Serum DBP was lower in the patients than in the controls (mean +/- SD 315 +/- 60 vs 371 +/- 44 mg/l, P less than 0.001). Serum DBP correlated positively with serum total protein, albumin, alpha 2-globulin, and the vitamin D metabolite levels in the patients. When correcting for differences in serum DBP, serum 25(OH)D and 24,25(OH)2D still were significantly lower in patients than in controls, whereas serum 1,25(OH)2D was not. The free 1,25(OH)2D index (10(5) x molar ratio 1,25(OH)2D/DBP) was lower in patients than in controls, but the level of significance was marginal. This difference was not significant when patients and controls with impairment of renal function were excluded. It is concluded that the difference in serum 25(OH)D and 24,25(OH)2D between patients and controls is largely preexistent. However, the lower serum 1,25(OH)2D in the patients is mainly caused by the trauma. The free 1,25(OH)2D concentrations are almost similar in the two groups when renal function is normal.

Interlaboratory Variation of Vitamin D Metabolite Measurements

Interlaboratory variation of the measurement of 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D by six laboratories in the Netherlands was studied. Three different serum samples and two different standard solutions of each metabolite were assayed. Substantial interlaboratory variation was found for the measurement of serum samples. The mean interlaboratory CVs for the 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D assays in the three sera were 48%, 38% and 20% respectively. The measurement of standard solutions of all metabolites showed relative little variation (mean CV 8%). The small number of samples allowed no evaluation of intralaboratory variation. The much higher CVs of the measurement of serum samples, when compared to standard solutions, may be attributed to differences in extraction and purification procedures which are probably responsible for the presence of varying amounts of interfering substances during the final quantification of metabolites.

Improvements in the Simultaneous Determination of Calcidiol and Calcitriol in Human Serum or Plasma

Technical improvements have been applied to reduce the time required for the determination of calcidiol and calcitriol in serum or plasma. The modifications include the use of Sep-Pak C18 cartridges for the extraction of calciol metabolites from serum instead of a classical liquid/liquid extraction, a considerable shortening of the HPLC purification time compared with our previously described method, and the application of HPLC with UV detection at 254 nm of calcidiol as an alternative to the usual competitive protein binding methods. In none of the 199 samples where calcidiol was determined by HPLC did we observe a detectable peak of ercalcidiol. Quantitation of calcidiol by HPLC and competitive protein binding was compared in 5 series of assays. The correlation between the two methods was 0.99. The average slope of the linear regression line when the HPLC values were plotted versus the competitive protein binding values was 1.14. The average intercept was 0.19 nmol/l. The mean within-run coefficient of variation for calcidiol in these series was 5% for the competitive protein binding method, and 4% for HPLC method. Between-run coefficients of variation were 6% and 12% for the competitive protein binding and for the HPLC method, respectively. Within-run and between-run coefficients of variation for calcitriol were 6% and 15%, respectively.