R. Barto

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects.

Abstract Background: Alterations in global DNA methylation are implicated in various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA. Methods: DNA was hydrolyzed using formic acid. Cytosine and 5-methylcytosine were separated by gradient-elution reversed-phase chromatography with a mobile phase containing nonafluoropentanoic acid (NFPA) as ion-pairing reagent and quantified using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers. Results: Linear calibration curves were obtained in the range 0.111–4.422 ng/μL [mean correlation co-efficient 0.9983 (SD=0.0011), n=9] for cytosine and 0.0048–0.1936 ng/μL [mean correlation coefficient 0.9991 (SD=0.0010), n=9] for 5-methylcytosine. The intra- and inter-assay CVs for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n=9) and 3.5% (n=8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n=6) and 6.5% (n=14), respectively for pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The limit of detection (signal-to-noise ratio 3) was 2 pg on-column for cytosine and 5-methylcytosine. In healthy subjects (n=109), the mCyt/tCyt ratio varied from 2.6% to 4.8% (median 4.1%). DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype (p=0.046). No association with B-vitamin status was observed. Conclusions: This LC-ESI-MS/MS method is easy to perform and offers reproducibility, selectivity and sensitivity for studying DNA methylation. The method allows a sample throughput of approximately 200 samples/week. The MTHFR C677T genotype influences age-related changes in DNA methylation. Clin Chem Lab Med 2007;45:903–11.

Estrogen Regulation of Intestinal Calcium Absorption in the Intact and Ovariectomized Adult Rat

Studies were carried out to examine the mechanism of action of estrogen on intestinal calcium absorption in the rat. Three‐month‐old Wistar rats were sham‐operated or ovariectomized (OVX). They were fed a diet containing 0.4% Ca, 0.4% P, and 2000 IU vitamin D3/kg. Eight weeks after operation, both OVX and sham‐operated rats were randomly assigned to eight treatment groups. Five groups received per 100 g of body weight 12.5 ng calcitriol (1,25‐dihydroxyvitamin D3); 7.5 μg of estradiol‐benzoate; 7.5 μg of estradiol‐benzoate and 0.1 mg of ICI 182780; 12.5 ng of calcitriol and 0.1 mg of ICI 182780; and 0.1 mg of ICI 182780, respectively. Three groups received the various vehicles used. Intestinal calcium absorption was measured in vivo using single pass perfusion of the duodenum. OVX did not change intestinal calcium absorption. A pharmacological dose of estradiol‐benzoate caused a significant increase in intestinal absorption of calcium, which was comparable to that of a pharmacological dose of calcitriol in both OVX and sham‐operated rats. Estrogen‐induced rise in intestinal calcium absorption was completely blocked to basal level by the pure estrogen receptor (ER) antagonist ICI 182780. In contrast, ICI 182780 did not antagonize calcitriol‐enhanced intestinal calcium absorption. Our findings suggest that estrogen stimulates intestinal calcium absorption via an ER.

Plasma choline and betaine correlate with serum folate, plasma S-adenosyl-methionine and S-adenosyl-homocysteine in healthy volunteers.

Abstract Background: Choline is essential for mammalian cell function. It plays a critical role in cell membrane integrity, neurotransmission, cell signaling and lipid metabolism. Moreover, choline is involved in methylation in two ways: a) its synthesis requires methyl groups donated by S-adenosyl-methionine (AdoMet); and b) choline oxidation product betaine methylates homocysteine (Hcy) to methionine (Met) and produces dimethylglycine. This later donates one carbon units to tetrahydrofolate (THF). Methods: To evaluate the correlations of choline and betaine with folate, AdoMet, S-anenosyl-homocysteine (AdoHcy), total homocysteine (tHcy), and DNA methylation, choline, betaine and dimethylglycine were measured by LC-MS/MS in plasma of 109 healthy volunteers, in whom folate, AdoMet, AdoHcy, tHcy, and DNA methylation have previously been reported. Results: Using a bivariate model, choline and betaine showed strong positive correlations with folate (r=0.346 and r=0.226), AdoHcy (r=0.468 and r=0.296), and correlated negatively with AdoMet/AdoHcy ratio (r=–0.246 and r=–0.379). Only choline was positively correlated with AdoMet (r=0.453). Using a multivariate linear regression model, choline correlated strongly with folate (β=17.416), AdoMet (β=61.272), and AdoHcy (β=9.215). Betaine correlated positively with folate (β=0.133) and negatively with tHcy (β=–0.194) ratio. Choline is an integral part of folate and methylation pathways. Conclusions: Our data highlight the importance of integrating choline in studies concerning addressing pathological conditions related to folate, homocysteine and methylation metabolism.

Oestrogen has no short‐term effect on intestinal strontium absorption in healthy postmenopausal women

Impaired intestinal calcium absorption in postmenopausal women is often indirectly linked to decreased serum 1,25(OH)2D or to intestinal resistance to its action rather than directly to low circulating oestrogen levels following the menopause. The purpose of this clinical study was to investigate the short‐term effect of oral 17β‐oestradiol on intestinal calcium absorption, with strontium as a marker.

Influence of Different Haemodialysis Modalities on AGE Peptide Levels: Intradialytic versus Long-Term Results

Background: Peptide-linked degradation products of advanced glycation end products (AGE peptides) accumulate in chronic haemodialysis (HD) patients and may contribute to a number of HD-related long-term complications, such as accelerated atherosclerosis. Methods: The influence of a single HD session versus long-term HD on serum AGE peptides was determined. The patients were randomized to HD with a low-flux polysulfone (PS; F 6HPS), a high-flux PS (F 60S), a superflux PS (F 500S), or a superflux cellulose triacetate (CTA; Tricea 150G) dialyzer. Results: During a single HD session, both AGE peptides and reference peptides decreased significantly (AGE peptides: Tricea 150G –37.0 ± 2.9%; F 6HPS –35.5 ± 2.4%; F 60S –39.5 ± 4.7%, and F 500S –43.3 ± 2.1%, p = 0.005; reference peptides: Tricea 150G –73.2 ± 8.8%; F 6HPS –73.2 ± 7.9%; F 60S –72.5 ± 8.2%, and F 500S –74.1 ± 7.3%, p = 0.005). After 12 weeks of HD with the superflux CTA, the AGE peptide levels decreased significantly (week 1: 2.7 ± 1.1 arbitrary units, week 12: 2.5 ± 1.2 arbitrary units, decrease 7.4%; p = 0.01), whereas the AGE peptide levels remained unchanged after HD with each of the other three modalities. The reference peptide levels did not change after 12 weeks of HD. Conclusion: Although AGE peptides can be effectively removed during a single HD session, superflux CTA seems to be the only modality capable of reducing AGE peptides in the long term.

Effect of estrogen on intestinal strontium absorption in postmenopausal women

OBJECTIVES there is considerable uncertainty about the underlying cause of decreased intestinal calcium absorption that occurs in postmenopausal women. In a previous study, estrogen treatment did not result in an increased intestinal calcium absorption using strontium as a marker. A possible explanation could be that the calcium/strontium load given to the women was too high ( approximately 600 mg Ca), which might result in an insensitive test with respect to the possible stimulation of active strontium transport by estrogen. Therefore, the purpose of this study was to reinvestigate the effect of estrogen on active intestinal strontium absorption using a load of 2.5 mmol of strontium only. METHODS the effect of estrogen on intestinal strontium absorption was measured in eight normal postmenopausal women. The study included two baseline strontium absorption tests, which were performed with an interval of 10 days for calculating the within subject variation (SER). Thereafter the effect of 2 months of estrogen treatment on intestinal strontium absorption was assessed. Fractional absorption (FC(240)) and the area under the concentration time curve (AUC) 4 h after an oral strontium load of 2.5 mmol were calculated. RESULTS the within subject SER of FC(240) and AUC(0-240) were 2.3+/-0.76 and 1.2+/-0.41, respectively. FC(240) and AUC(0-240) of strontium were unchanged after treatment with estrogen. CONCLUSIONS in normal postmenopausal women, we did not find a modulating effect of short-term treatment with a (supra) physiological dose of estrogen on intestinal calcium absorption as measured by the strontium absorption test.

Topically applied low-dose calcitriol has no calciotropic effect in patients with stable plaque psoriasis

BACKGROUND Topical calcitriol, a potent inhibitor of cell proliferation and inducer of terminal cell differentiation, can clear psoriasis. However, possible side effects on calcium and bone metabolism from transdermal absorption have not been evaluated. OBJECTIVE The calciotropic effects of low-dose calcitriol (3 micrograms/gm) ointment, applied twice daily for 6 weeks, were investigated. METHODS A double-blind study was carried out in 18 patients with chronic stable plaque-type psoriasis, of whom nine were treated with calcitriol (3 micrograms/gm) and nine with betamethasone dipropionate (500 micrograms/gm). The main end points were calcitriol plasma concentrations, intestinal calcium absorption, and bone turnover. RESULTS Serum alkaline phosphatase concentrations increased slightly (p < 0.02) and intestinal calcium absorption decreased slightly (p < 0.01) in the calcitriol-treated group. However, the alterations were too small to have any clinical relevance. CONCLUSION Low-dose calcitriol, topically applied for 6 weeks on a maximal body surface area of 30%, can be considered as safe regarding calcium and bone metabolism.

Human Pharmacokinetics of Orally Administered (24 R)-Hydroxycalcidiol

To gain an insight in the regulation of (24R)-hydroxycalcidiol, we studied the pharmacokinetics of orally administered (24R)-hydroxycalcidiol in 6 healthy subjects without calcium supplementation, in 4 healthy subjects with calcium supplementation and in 6 patients with primary hyperparathyroidism. Various quantities related to calcium and vitamin D metabolism were also monitored. In the healthy subjects without calcium supplementation, the basal (24R)-hydroxycalcidiol concentration (Cb) in serum was 2.4 +/- 0.8 nmol/l (mean +/- SD, n = 5), the terminal serum half-time (t 1/2) 7.2 +/- 1.4 days, the production rate 0.05 +/- 0.01 nmol/kg.day, and the production rate/[calcidiol] ratio (1.5 +/- 0.4 x 10(-3) l/kg.day). In the healthy subjects studied, the serum concentration vs time curves exhibited a second maximum after administration, possibly due to binding by intestinal cells or (partial) uptake by the lymph system. In the calcium-supplemented healthy subjects, the pharmacokinetic quantities were not significantly different while the area under the serum concentration-time curve and the estimated bioavailability were significantly decreased. Basal concentration (Cb), production rate and the production rate/[calcidiol] ratio were significantly lower in patients with primary hyperparathyroidism but t 1/2 was unchanged. Exogenous (24R)-hydroxycalcidiol had no clear effect on calcium and vitamin D metabolism. In conclusion, a) exogenous (24R)-hydroxycalcidiol has no clear effect on calcium and vitamin D metabolism, b) clearance and production rate of (24R)-hydroxycalcidiol are not affected by calcium supplementation, c) bioavailability is lower in the calcium-supplemented state, d) basal concentration (Cb) and production rate are significantly decreased in patients with hyperparathyroidism.