Cholesterol Interference in the Assessment of Vitamin D Status: A Canadian Health Measures Survey Biobank Project.

BACKGROUND: Matrix effects are a known problem with immunoassays measuring serum 25-hydroxyvitamin D [25(OH)D]. OBJECTIVES: To determine if the inverse association between serum 25(OH)D and serum cholesterol concentrations is a function of assay method: Diasorin Liaison 25(OH) Vitamin D Total Assay (Liaison Total Assay), an immunoassay, compared with liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Canadian Health Measures Survey data and biobank serum (White males aged 20-79 y, n = 392) were evaluated for bias in serum 25(OH)D using Bland-Altman plots. Differences in serum 25(OH)D (Liaison Total Assay - LC-MS/MS) were compared among non-HDL-cholesterol <4.2 (n = 295) compared with ≥4.2 (n = 97) mmol/L and total cholesterol groups <5.2 (n = 256) compared with ≥5.2 (n = 136) mmol/L, and associations tested between 25(OH)D and non-HDL-cholesterol or total cholesterol concentrations, using regression. RESULTS: Serum 25(OH)D measured using Liaison Total Assay ranged from 10.7 to 137.0 nmol/L and 14.4 to 137.9 nmol/L by LC-MS/MS. Liaison Total Assay - LC-MS/MS showed a negative bias of 5.5 (95% limits of agreement -23.8, 12.7) nmol/L. Differences in 25(OH)D were -4.0 ± 9.0 (±SD) nmol/L if non-HDL-cholesterol was <4.2 mmol/L and -10.2 ± 8.7 nmol/L if ≥4.2 mmol/L (P < 0.0001). Differences in 25(OH)D, if total cholesterol was <5.2 mmol/L, were -3.4 ± 8.6 nmol/L and -9.6 ± 9.3 nmol/L if ≥5.2 mmol/L (P < 0.0001). Serum non-HDL-cholesterol (beta -3.17, P = 0.0014) and total cholesterol (beta -2.77, P = 0.0046) were inversely associated with Liaison Total Assay 25(OH)D (adjusted for age, fasting, and body mass index), but not with LC-MS/MS measured 25(OH)D. Interference by these lipoproteins was not eliminated by standardization of the Liaison Total Assay. Similar associations were observed with triglycerides as for the lipoproteins. CONCLUSIONS: Total cholesterol inversely associates with 25(OH)D, which is likely due to elevated non-HDL-cholesterol lipoprotein or triglyceride interference with the Liaison Total Assay. This is important as elevated cholesterol is common, and an underestimation of vitamin D status could be an unnecessary cause for concern.

Low 25-hydroxyvitamin D levels are more prevalent in Canadians of South Asian than European ancestry inhabiting the National Capital Region of Canada.

The US Institute of Medicine defined serum 25-hydroxyvitamin D (25OHD) cut point values of 30 nmol/L and 40 nmol/L were used to assess the vitamin D status of South Asian and European Canadians of self-identified ancestry living in the National Capital Region of Canada. Serum 25OHD values were measured in the spring and fall of 2012 to represent status during the winter and summer months, respectively. A total of 1238 measurements were obtained from 669 participants (49% South Asian ancestry): some participants were measured only once (spring or fall). Median 25OHD values were significantly higher in participants of European ancestry: 70.8 nmol/L (68.1, 73.5; 95% CI) versus South Asian ancestry: 42.7 nmol/L (40.5, 45.0; P<0.001). Spring vs. fall differences were small for each ethnic group and significant only for those of European ancestry (2.9, CI: 1.0-4.9 nmol/L; P = 0.01). Among participants of South Asian ancestry, 27.3% (fall) and 29.1% (spring) of females had values <40 nmol/L while the percentages for males were considerably higher (36.5% and 44.2%, respectively). The corresponding values for participants of European ancestry were ≤10%, showing that the South Asian participants were less likely to achieve the 25OHD concentrations established by the IOM for optimum bone health. Investigation of the factors related to serum 25OHD levels showed that supplement intake and ethnic background were associated with the biggest differences. Skin color was not a major factor, suggesting that genetic factors are responsible for the observed differences between participants of different ethnic backgrounds.

Fermentable Carbohydrates Differentially Affect Colon Tumor Formation in Azoxymethane-Induced Male Fischer 344 Rats.

BACKGROUND: The role of fermentation compared with the source or type of the fermentable material in colon tumorigenesis remains an issue in refining the definition of dietary fiber (DF). OBJECTIVE: The aim of this study was to investigate the fermentation and source-specific effects of various carbohydrates in a medium-term colon tumorigenesis model. METHODS: Six-week-old male Fischer 344 rats were randomly allocated into 6 groups (n = 36/group) to receive either AIN-93G (control) or diets containing fructooligosaccharides, wheat bran (WB), oat bran (OB), polydextrose, or high-amylose maize starch (HAMS), each adjusted to contain a total DF concentration of 7% (wt:wt) and have a fermentability of 3% (wt:wt). After 2 wk, 24 rats/group received 2 subcutaneous doses of azoxymethane (at 15 mg/kg body weight) 1 wk apart while 12 rats/group were injected with a saline vehicle; all rats were maintained on the assigned diets for 24 wk postinjection and then killed. Colon tumor outcomes and pathology together with cecal short-chain fatty acid composition were assessed. RESULTS: No tumors were found in saline-injected rats, and all subsequent analyses were restricted to azoxymethane-injected rats. Colon tumor incidence was significantly lower in the polydextrose (21%) and WB (13%) groups than in the control group (63%; P < 0.05) but not different from the fructooligosaccharide (58%), HAMS (46%), and OB (33%) groups. In comparison to the control group (8 proximal/31 total tumors), fermentable materials reduced the number of tumors (P < 0.05) originating in the proximal colon: HAMS (5/15), polydextrose (2/7), OB (2/9), fructooligosaccharides (1/21), and WB (1/3). The mean ± SEM number of tumors/tumor-bearing rats was significantly lower in the WB (1.00 ± 0.00), OB (1.13 ± 0.13), and HAMS (1.36 ± 0.15) groups than in the control group (2.07 ± 0.27; P < 0.02); other groups did not differ. The mean ± SEM tumor burden/diet group was lower in the WB (1.2 ± 0.7 mm2), polydextrose (6.7 ± 3.2 mm2), and OB (7.0 ± 3.0 mm2) groups than in the control (21.4 ± 5.9 mm2) and fructooligosaccharide (22.1 ± 7.1 mm2; P < 0.05) groups but not significantly different from the HAMS group (15.1 ± 6.1 mm2). Total cecal SCFA concentrations did not differ among diet groups (overall mean ± SEM: 81 ± 4 µmol/g wet weight). CONCLUSION: The rate and extent of fermentation of the carbohydrate material as well as the characteristics of the material in the lumen of the lower gastrointestinal tract all appear to have an important role in tumor outcomes in the azoxymethane-induced rat colon tumorigenesis assay.

The Influence of Euthanasia Methods on Rat Liver Metabolism.

An important consideration in any terminal experiment is the method for euthanizing animals. Although the prime consideration is that the method is humane, some methods can have a dramatic impact on experimental outcomes. To determine the optimal method of euthanasia in metabolic experiments, a physical method (decapitation), two asphyxiation methods (CO2 and O2/CO2), and anesthetic (isoflurane) exposure followed by exsanguination were compared for their effects on liver metabolism. Changes in metabolism were monitored by following the activities of several key metabolic enzymes that are known to be susceptible to alterations in extracellular hormones as well as to changes in intracellular energy availability. The substrates and products of these enzymes also were monitored to better estimate their in vivo activity. Decapitated animals were used as the baseline for all comparisons. The results showed that euthanasia after exposure to 3 min isoflurane, euthanasia by exposure to a pure CO2 atmosphere for 2.5 min (CO2), and euthanasia by exposure to 1 min pure O2 followed by 2.5 min CO2 (O2/CO2) stimulated the enzymes involved in glycogen breakdown and glucose utilization. After CO2 or O2/CO2 asphyxiation, liver glycogen stores fell to approximately one-half those in the decapitated animals. No significant losses in liver glycogen were apparent after exsanguination under isoflurane anesthesia. In addition, differences between euthanasia methods were noted when the pattern of enzyme activity was compared: enzymes at the start of the glycolytic pathway were stimulated after CO2 or O2/CO2 euthanasia, but the terminal glycolytic enzyme was stimulated only after O2/CO2 euthanasia. Euthanasia by CO2 or O2/CO2 methods significantly decreased the regulatory enzyme of branched-chained amino acid degradation. This study clearly indicates that the method of euthanasia can have a dramatic impact on experimental data and, in particular, on liver metabolism.