FSMP-06. IN VIVO MONITORING OF LDHA EXPRESSION IN GLIOBLASTOMA USING QUANTITATIVE EXCHANGED-LABEL TURNOVER 1H MRS TECHNIQUE

Most cancers, including glioblastomas (GBMs), rely extensively on glycolysis to support growth, proliferation, and survival. A hallmark of this elevated glycolysis is overexpression of Lactate dehydrogenase-A (LDHA) protein leading to increased uptake of glucose and overproduction of lactate. Various clinical trials using LDHA as a target for diagnosis and treatment have yielded encouraging results. However, in vivo monitoring of LDHA expression has been challenging due to either requirement of administration of radioactive substrates or specialized hardware. In this presentation, we will demonstrate a new method-quantitative exchanged-label turnover MRS (QELT, or simply qMRS)-that increases the sensitivity of magnetic resonance-based metabolic mapping without the requirement for specialized hardware. qMRS relies on the administration of deuterated (2H-labeled) substrates to track the production of downstream metabolites. Since 2H is invisible on 1H MRS, replacement of 1H with 2H due to metabolic turnover leads to an overall reduction in 1H MRS signal for the corresponding metabolites. We applied our qMRS technique to monitor the rate of lactate production in a preclinical GBM model. Infusion of [6,6’-2H2]glucose led to downstream deuterium labeling of lactate, thereby resulting in a reduction in the 1.33 ppm lactate-CH3 peak on 1H MRS over time. The subtraction of post-administration 1H MR spectra from the pre-infusion spectra aided in the determination of the kinetics of the lactate turnover. We believe that the detection and quantification of lactate production kinetics may provide crucial information regarding tumor LDHA expression non-invasively in GBMs without requiring biopsies. Hence, qMRS is expected to open up new opportunities to probe LDHA expression differences in a variety of gliomas, including GBMs and astrocytomas. This method takes advantage of the universal availability and ease of implementation of 1H MRS on all clinical and preclinical magnetic resonance scanners.

Estimation of gluconeogenesis in newborn infants

The rate of glucose turnover (Ra) and gluconeogenesis (GNG) via pyruvate were quantified in seven full-term healthy babies between 24 and 48 h after birth and in twelve low-birth-weight infants on days 3 and 4 by use of [13C6]glucose and2H2O. The preterm babies were receiving parenteral alimentation of either glucose or glucose plus amino acid with or without lipids. The contribution of GNG to glucose production was measured by the appearance of 2H on C-6 of glucose. Glucose Ra in full-term babies was 30 ± 1.7 (SD) μmol · kg−1 · min−1. GNG via pyruvate contributed ∼31% to glucose Ra. In preterm babies, the contribution of GNG to endogenous glucose Rawas variable (range 6–60%). The highest contribution was in infants receiving low rates of exogenous glucose infusion. In an additional group of infants of normal and diabetic mothers, lactate turnover and its incorporation into glucose were measured within 4–24 h of birth by use of [13C3]lactate tracer. The rate of lactate turnover was 38 μmol · kg−1 · min−1, and lactate C, not corrected for loss of tracer in the tricarboxylic acid cycle, contributed ∼18% to glucose C. Lactate and glucose kinetics were similar in infants that were small for their gestational age and in normal infants or infants of diabetic mothers. These data show that gluconeogenesis is evident soon after birth in the newborn infant and that, even after a brief fast (5 h), GNG via pyruvate makes a significant contribution to glucose production in healthy full-term infants. These data may have important implications for the nutritional support of the healthy and sick newborn infant.

Proliferative activity and tumorigenic conversion: impact on cellular metabolism in 3-D culture

Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of[Formula: see text] distributions with microelectrodes, and determination of cellular oxygen uptake rates (Q˙[Formula: see text]) have been applied. In the ras-transfected, tumorigenic spheroid types, the size dependencies of some of the measured parameters exhibited sharp breaks at diameters of ∼830 μm for Rat1-T1 and ∼970 μm for MR1 spheroids, respectively, suggesting that some fundamental change in cell metabolism occurred at these characteristic diameters (denoted as “metabolic switch”).Q˙[Formula: see text]decreased and lactate concentration increased as functions of size below the characteristic diameters. Concomitantly, glucose and lactate turnover rates decreased in MR1 spheroids and increased in Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting cell quiescence and lactate accumulation) showed an enhancement ofQ˙[Formula: see text]with size. Systematic variations in the ATP and glucose levels in the viable cell rim were observed for Rat1-T1 spheroids only. Proliferative activity, Q˙[Formula: see text], and ATP levels in small, nontumorigenic Rat1 and M1 aggregates did not differ systematically from those recorded in the largest spheroids of the corresponding ras transfectants. Unexpectedly, respiratory activity was present not only in viable but also in the morphologically disintegrated core regions of M1 aggregates. Our data suggest that myc but not rastransfection exerts major impacts on cell metabolism. Moreover, some kind of switch has been detected that triggers profound readjustment of tumor cell metabolism when proliferative activity begins to stagnate, and that is likely to initiate some other, yet unidentified energy-consuming process.

Lactate and pyruvate isotopic enrichments in plasma and tissues of postabsorptive and starved rats

It has been proposed that plasma pyruvate isotopic enrichment (IE) during infusion of labeled lactate could be used to estimate the intracellular IE of lactate and pyruvate and thus to calculate their turnover rate. We determined the relations of plasma and tissue IE of lactate and pyruvate in anesthetized rats infused with [3-13C]lactate in an artery and sampled from a vein (A-V mode) or infused in a vein and sampled from an artery (V-A mode). In both groups of rats, the ratio of tissue to plasma lactate IE was < 1 with large differences between tissues: the highest ratio was observed in heart and the lowest in soleus. With the exception of liver, this ratio was higher in the A-V than in the V-A mode. Pyruvate IE was lower than lactate IE in tissues, with a few exceptions, and in plasma. This ratio of pyruvate to lactate IE was approximately 0.70 in plasma in A-V and V-A modes. Moreover pyruvate IE was also always higher in plasma than in tissues. This seemingly surprising result could be explained by the production of labeled pyruvate from labeled lactate inside the circulation by erythrocytes, because we observed a rapid isotopic equilibrium between lactate and pyruvate in blood "in vitro." Apparent lactate turnover was higher in the A-V than in the V-A mode when it was calculated using lactate as well as pyruvate IE. Therefore plasma pyruvate IE cannot be used in rats to estimate tissue IE and did not reconcile turnover rates measured using the A-V or V-A mode.(ABSTRACT TRUNCATED AT 250 WORDS)