Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites.

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Studies on the energy metabolism of opossum (Didelphis Virginiana) erythrocytes. I. Utilization of carbohydrates and purine nucleosides.

Opossum erythrocytes filtered through cellulose columns were used to estimate their permeability to D-glucose and optimum inorganic phosphate requirement for D-glucose utilization at pH 7.4 and 8.1. D-Glucose readily penetrated opossum red cells; there was no measurable difference whether plasma or electrolyte solution served as the suspending medium. Optimum extracellular inorganic phosphate concentration for glucose utilization as indicated by red cell lactate production was pH-dependent, with a sharp optimum of 30 mmol/liter at pH 8.1. Whereas glucose, fructose, mannose, dihydroxyacetone, adenosine, and inosine were readily utilized at pH 7.4 and Pi 30 mmol/liter as shown by net lactate and ATP production by the red cells, galactose and ribose as substrates were not metabolized. In electrolyte, Pi 30 mmol/liter, and pH 7.4 glucose utilization by opossum red cells averaged 3.5 mumol, at pH 8.1, 9.5 mumol/ml cells/hr were utilized. Red cells suspended in leukocyte-free plasma utilized D-glucose at a rate of 3.0 mumol/ml/hr at pH 7.5. Seven percent of D-glucose flowed through the pentose phosphate pathway; this rate increased 11-fold by methylene blue stimulation. The amount of D-glucose recycled through the pentose phosphate pathway increased 300-fold in the presence of the redox dye.

Cow red blood cells. III. Postnatal adaptation of energy metabolism in the calf red blood cells.

1. The change in energy metabolism of red blood cells from the newborn calf to adult cow was examined utilizing a number of metabolic substrates including glyceraldehyde, dihydroxyacetone, ribose, glucose, adenosine and inosine. 2. All of these substrates are utilizes by the newborn calf cells to a varying degree. With glyceraldehyde, dihydroxyacetone or glucose as a substrate, lactate is formed at a rate of 2-3 mumol/ml cells per h. As in other species, ribose utilization depends on substrate concentration, with an optimum of 3 mM ribose yielding lactate 1-1.5 mumol/ml cells per h in the calf cells. 3. In sharp contrast, adult cow red blood cells lost the bulk of the postnatal metabolic substrate affinities except for glyceraldehyde and glucose which are consumed at less than half of the rate at birth. 4. While the transition of the metabolic properties from the newborn to the adult state takes place within 2 to 3 months after birth, the red blood cells produced shortly after birth have already assumed the metabolic machinery characteristic to the adult cells. 5. Even though adenosine in itself is a poor substrate in producing lactate, a net synthesis of ATP from adenosine can take place in both calf and cow cells provided that an alternate carbon source such as glyceraldehyde, dihydroxyacetone or glucose is given. 6. Of the test substrates, glucose is the only substrate for the adult cow cells exhibiting a greater than 50% increase in utilization by exogenously added adenine. By contrast, the calf cell is affected to a much lesser extent. The possible in vivo regulatory metabolic role of certain purine and pyrimidine compounds unique to the adult stage of this species is discussed.