Ana Fagulha

Lack of association of vitamin D receptor gene polymorphisms with susceptibility to type 1 diabetes mellitus in the Portuguese population.

The vitamin D receptor (VDR) gene is a candidate gene for susceptibility to autoimmune disorders. Association studies of VDR polymorphisms and risk of type 1 diabetes often produced conflicting results in different ethnic backgrounds. The aim of this study was to test for association between common VDR polymorphisms and the genetic susceptibility to type 1 diabetes in the Portuguese population. We genotyped 207 patients with type 1 diabetes and 249 controls for the FokI T>C (rs10735810), BsmI A>G (rs1544410), ApaI G>T (rs7975232), and TaqI C>T (rs731236) single nucleotide polymorphisms by polymerase chain reaction and restriction fragment length polymorphism analysis. The distribution of VDR genotype, allele, and haplotype frequencies did not differ significantly between patients and controls. These data suggest that the single nucleotide polymorphisms of the VDR gene are unlikely to contribute significantly to type 1 diabetes susceptibility in the Portuguese population.

NMR Derivatives for Quantification of 2H and 13C‐Enrichment of Human Glucuronide from Metabolic Tracers

Quantification of 2H and 13C enrichment distributions in human urinary glucuronide following ingestion of 2H2O and 13C gluconeogenic tracers was achieved by NMR spectroscopy of the 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone and 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivatives. The derivatization process is simple and can be applied to any glucuronide species. The derivatives are highly soluble in acetonitrile and generate well‐resolved and narrow 2H and 13C NMR signals. The 1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided resolution of the six glucuronide 13C signals and numerous 13C isotopomer populations through one‐ and two‐bond 13C‐13C‐coupling, while the 5‐O‐acetyl‐1,2‐O‐isopropylidene‐α‐D‐glucofuranurono‐6,3‐lactone derivative provided complete resolution of the 2H NMR signals for the five glucuronide hydrogens. The isopropylidene methyl signals were also resolved and provided an internal 2H enrichment standard following the acetonation of glucuronolactone with deuterated acetone.

Noninvasive Analysis of Hepatic Glycogen Kinetics Before and After Breakfast with Deuterated Water and Acetaminophen

The contributions of hepatic glycogenolysis to fasting glucose production and direct pathway to hepatic glycogen synthesis were quantified in eight type 1 diabetic patients and nine healthy control subjects by ingestion of 2H2O and acetaminophen before breakfast followed by analysis of urinary water and acetaminophen glucuronide. After overnight fasting, enrichment of glucuronide position 5 relative to body water (G5/body water) was significantly higher in type 1 diabetic patients compared with control subjects, indicating a reduced contribution of glycogenolysis to glucose production (38 ± 3 vs. 46 ± 2%). Following breakfast, G5/body water was significantly higher in type 1 diabetic patients, indicating a smaller direct pathway contribution to glycogen synthesis (47 ± 2 vs. 59 ± 2%). Glucuronide hydrogen 2 enrichment (G2) was equivalent to body water during fasting (G2/body water 0.94 ± 0.03 and 1.02 ± 0.06 for control and type 1 diabetic subjects, respectively) but was significantly lower after breakfast (G2/body water 0.78 ± 0.03 and 0.82 ± 0.05 for control and type 1 diabetic subjects, respectively). The reduced postprandial G2 levels reflect incomplete glucose-6-phosphate–fructose-6-phosphate exchange or glycogen synthesis from dietary galactose. Unlike current measurements of human hepatic glycogen metabolism, the 2H2O/acetaminophen assay does not require specialized on-site clinical equipment or personnel.

Simple measurement of gluconeogenesis by direct 2H NMR analysis of menthol glucuronide enrichment from 2H2O

The contribution of gluconeogenesis to fasting glucose production was determined by a simple measurement of urinary menthol glucuronide (MG) 2H enrichment from 2H2O. Following ingestion of 2H2O (0.5% body water) during an overnight fast and a pharmacological dose (400 mg) of a commercial peppermint oil preparation the next morning, 364 μmol MG was quantitatively recovered from a 2‐h urine collection by ether extraction and a 125 μmol portion was directly analyzed by 2H NMR. The glucuronide 2H‐signals were fully resolved and their relative intensities matched those of the monoacetone glucose derivative. The pharmacokinetics and yields of urinary MG after ingestion of 400 mg peppermint oil as either gelatin or enteric‐coated capsules 1 h before breakfast were quantified in five healthy subjects. Gelatin capsules yielded 197 ± 81 μmol of MG from the initial 2‐h urine collection while enteric‐coated capsules gave 238 ± 84 μmol MG from the 2‐ to 4‐h urine collection. Magn Reson Med 54:429–434, 2005.

Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects

During feeding, dietary galactose is a potential source of hepatic glycogen synthesis; but its contribution has not been measured to date. In the presence of deuterated water ((2)H(2)O), uridine diphosphate (UDP)-glucose derived from galactose is not enriched, whereas the remainder derived from glucose-6-phosphate (G6P) is enriched in position 2 to the same level as body water, assuming complete G6P-fructose-6-phosphate (F6P) exchange. Hence, the difference between UDP-glucose position 2 and body water enrichments reflects the contribution of galactose to glycogen synthesis relative to all other sources. In study 1, G6P-F6P exchange in 6 healthy subjects was quantified by supplementing a milk-containing breakfast meal with 10 g of [U-(2)H(7)]glucose and quantifying the depletion of position 2 enrichment in urinary menthol glucuronide. In study 2, another 6 subjects ingested (2)H(2)O and acetaminophen followed by an identical breakfast meal with 10 g of [1-(13)C]glucose to resolve direct/indirect pathways and galactose contributions to glycogen synthesis. Metabolite enrichments were determined by (2)H and (13)C nuclear magnetic resonance. In study 1, G6P-F6P exchange approached completion; therefore, the difference between position 2 and body water enrichments in study 2 (0.20% ± 0.03% vs 0.27% ± 0.03%, P < .005) was attributed to galactose glycogenesis. Dietary galactose contributed 19% ± 3% to glycogen synthesis. Of the remainder, 58% ± 5% was derived from the direct pathway and 22% ± 4% via the indirect pathway. The contribution of galactose to hepatic glycogen synthesis was resolved from that of direct and indirect pathways using a combination of (2)H(2)O and [1-(13)C]glucose tracers.

Sources of hepatic glucose production by 2H2O ingestion and Bayesian analysis of 2H glucuronide enrichment

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after 2H2O ingestion by Bayesian analysis of the position 2 and 5 2H‐NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 ± 1.0 kg/m2; fasting glucose = 4.7 ± 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 ± 0.7 kg/m2; fasting glucose = 7.1 ± 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% ± 5%. This uncertainty was associated with a mean signal‐to‐noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% ± 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% ± 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% ± 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia. Magn Reson Med 60:517–523, 2008.