Carla Baptista

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 during an oral glucose tolerance test: Effect of transaldolase exchange on flux estimates

Sources of hepatic glycogen synthesis during an oral glucose tolerance test were evaluated in six healthy subjects by enrichment of a 75‐g glucose load with 6.67% [U‐13C]glucose and 3.33% [U‐2H7]glucose and analysis of plasma glucose and hepatic uridine diphosphate–glucose enrichments (sampled as urinary menthol glucuronide) by 2H and 13C nuclear magnetic resonance. The direct pathway contribution, as estimated from the dilution of [U‐13C]glucose between plasma glucose and glucuronide, was unexpectedly low (36 ± 5%). With [U‐2H7]glucose, direct pathway estimates based on the dilution of position 3 2H‐enrichment between plasma glucose and glucuronide were significantly higher (51 ± 6%, P = 0.05). These differences reflect the exchange of the carbon 4, 5, and 6 moiety of fructose‐6‐phosphate and glyceraldehyde‐3‐phosphate catalyzed by transaldolase. As further evidence of this exchange, 2H‐enrichments in glucuronide positions 4 and 5 were inferior to those of position 3. From the difference in glucuronide positions 5 and 3 enrichments, the fraction of direct pathway carbons that experienced transaldolase exchange was estimated at 21 ± 4%. In conclusion, the direct pathway contributes only half of hepatic glycogen synthesis during an oral glucose tolerance test. Glucose tracers labeled in positions 4, 5, or 6 will give significant underestimates of direct pathway activity because of transaldolase exchange. Magn Reson Med, 2009.

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.

Kidney transplantation and diabetes: Posttransplantation malignancy

THE DEVELOPMENT of de novo malignancies in kidney transplant recipients represents a major problem. The reported prevalence is between 1% and 16% in Western countries and 1% to 3% in Asian countries. In our Center, the overall prevalence of 7.13% was reported in 1997 by Arnaldo et al. The most prevalent cancers are squamous cell carcinomas and basal cell carcinomas of the skin with an increasing cumulative incidence with longer survival of the patient. Epidemiological data show several risk factors, including pigmentary characteristics, solar irradiation, viral warts, type and doses of long-term immunosuppressants, presence of an oncogenic virus (EpsteinBarr), with age, with time of dialysis, genetic factors and diabetes among other considerations. The posttransplantation lymphoproliferative diseases, lymphoma, and Kaposi’s sarcoma are associated with the immunosuppressive therapy and oncogenic viruses. Type 2 diabetes mellitus (DM2) has been related to the risk for colon, endometrial, pancreatic, and hepatic cancers as well as an increased risk of non-Hodgkin’s lymphoma. Until now there are no reports of similar correlations with type 1 diabetes mellitus (DM1). Posttransplantation diabetes mellitus is in some ways similar to DM2, including insulin resistance and the presence of an overweight or obesity situation, but there are no reports on the prevalence of malignancy in this group of patients. The aim of this study was to determine the prevalence of malignancy during follow-up of all patients with diabetes bearing a kidney transplant in our Center.