Optimized methods to measure acetoacetate, 3‐hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

Stappenbeck, R.; Hodson, A. W.; Skillen, A. W.; Agius, Loranne; Alberti, K. G. M. M.

doi:10.1155/s1463924690000281

Cited by 18 publications

(12 citation statements)

References 3 publications

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“…Cortisol (22), GH (23), free insulin (24), and C peptide were measured using RIAs. Glycerol was measured using an enzymatic fluorometric method (25), and NEFA (Wako kit from Alpha Laboratories, Eastleigh, UK) and BOB (Randox, Crumlin, Ireland) were measured using enzymatic methods. Paired samples were run in single assays.…”

Section: Methodsmentioning

confidence: 99%

Reduced Counterregulation during Hypoglycemia with Raised Circulating Nonglucose Lipid Substrates: Evidence for Regional Differences in Metabolic Capacity in the Human Brain?

Evans

Matyka

Lomas

et al. 1998

The Journal of Clinical Endocrinology &Amp; Metabolism

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We have investigated the potential for the human brain to use lipid fuels during acute hypoglycemia. Nine healthy male subjects underwent hyperinsulinemic (1.5 mU/kg x min) stepped hypoglycemic clamps on two occasions, infusing Intralipid (20%) and heparin (0.1 U/kg x min) on one occasion only (ILH), with an identical study without infusion of ILH acting as a control. Five subjects also underwent euglycemic clamping with Intralipid/heparin infusion. During hypoglycemia, ILH raised circulating levels of nonesterified fatty acids, glycerol, and beta-hydroxybutyrate, although the latter did not rise until after the onset of counterregulation. With ILH, epinephrine responses [area under the curve (AUC), 127.9 +/- 31.7 vs. 175.1 +/- 27.4 nmol/L x 180 min; P = 0.03] and GH responses (AUC, 260 +/- 91 vs. 1009 +/- 150, P < 0.01) were reduced and delayed (glucose thresholds, 2.8 +/- 0.04 vs. 3.0 +/- 0.1 mmol/L; P = 0.04), with a trend toward reduced cortisol responses. Similarly, hypoglycemic symptom scores were diminished during ILH (AUC, 647 +/- 162 vs. 1222 +/- 874; P = 0.03). However, there was no significant effect on the deterioration in four-choice reaction time, one measure of cognitive deterioration [glucose thresholds, 2.6 +/- 0.1 vs. 2.7 +/- 0.1 mmol/L, ILH vs. control (P = 0.75); AUC, 1420 +/- 710 vs. 2250 +/- 1080 ms/min (P = 0.59)]. During euglycemic clamping with Intralipid/heparin infusion studies, there was no rise in hormones, four-choice reaction time, or symptoms other than hunger and tiredness. Both nonesterified fatty acids and glycerol can penetrate the mammalian brain and be metabolized. Raised levels were able to reduce neurohumoral responses to hypoglycemia, but could not protect cognitive function. This suggests that regional differences exist in human brain metabolism between glucose-sensing and cognitive areas of brain, which may be important in the understanding of the mechanisms of glucose sensing and in the genesis of hypoglycemia unawareness in insulin-dependent diabetes.

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Section: Methodsmentioning

confidence: 99%

Reduced Counterregulation during Hypoglycemia with Raised Circulating Nonglucose Lipid Substrates: Evidence for Regional Differences in Metabolic Capacity in the Human Brain?

Evans

Matyka

Lomas

et al. 1998

The Journal of Clinical Endocrinology &Amp; Metabolism

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“…1, D-F). Glucose was determined by the hexokinase, glucose 6-phosphate dehydrogenase-coupled assay (56). Production of pyruvate plus lactate or glucose is expressed as nmol/2 h/mg of protein, and glucose production is also expressed as a percentage of total production of pyruvate ϩ lactate ϩ glucose (expressed as C3 units).…”

Section: Metabolite Assaysmentioning

confidence: 99%

Low metformin causes a more oxidized mitochondrial NADH/NAD redox state in hepatocytes and inhibits gluconeogenesis by a redox-independent mechanism

Alshawi

Agius

2019

Journal of Biological Chemistry

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The mechanisms by which metformin (dimethylbiguanide) inhibits hepatic gluconeogenesis at concentrations relevant for type 2 diabetes therapy remain debated. Two proposed mechanisms are 1) inhibition of mitochondrial Complex 1 with consequent compromised ATP and AMP homeostasis or 2) inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) and thereby attenuated transfer of reducing equivalents from the cytoplasm to mitochondria, resulting in a raised lactate/pyruvate ratio and redox-dependent inhibition of gluconeogenesis from reduced but not oxidized substrates. Here, we show that metformin has a biphasic effect on the mitochondrial NADH/ NAD redox state in mouse hepatocytes. A low cell dose of metformin (therapeutic equivalent: <2 nmol/mg) caused a more oxidized mitochondrial NADH/NAD state and an increase in lactate/pyruvate ratio, whereas a higher metformin dose (>5 nmol/mg) caused a more reduced mitochondrial NADH/NAD state similar to Complex 1 inhibition by rotenone. The low metformin dose inhibited gluconeogenesis from both oxidized (dihydroxyacetone) and reduced (xylitol) substrates by preferential partitioning of substrate toward glycolysis by a redoxindependent mechanism that is best explained by allosteric regulation at phosphofructokinase-1 (PFK1) and/or fructose 1,6-bisphosphatase (FBP1) in association with a decrease in cell glycerol 3-phosphate, an inhibitor of PFK1, rather than by inhibition of transfer of reducing equivalents. We conclude that at a low pharmacological load, the metformin effects on the lactate/ pyruvate ratio and glucose production are explained by attenuation of transmitochondrial electrogenic transport mechanisms with consequent compromised malate-aspartate shuttle and changes in allosteric effectors of PFK1 and FBP1.

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“…Glucose, 3‐hydroxybutyrate, glycerol and acetoacetate were measured enzymatically following the procedure described by Stappenbeck et al . [10].…”

Section: Methodsmentioning

confidence: 99%

“…NEFA concentrations were measured using a commercially available enzymatic assay (Wako NEFA-C, Neuss, Germany). Glucose, 3-hydroxybutyrate, glycerol and acetoacetate were measured enzymatically following the procedure described by Stappenbeck et al [10].…”

Section: Laboratory Analysesmentioning

confidence: 99%

Effects of gemfibrozil on insulin resistance to fat metabolism in subjects with type 2 diabetes and hypertriglyceridaemia

Whitelaw¹,

Smith²,

Nattrass³

2002

Diabetes Obesity Metabolism

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Optimized methods to measure acetoacetate, 3‐hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

Cited by 18 publications

References 3 publications

Reduced Counterregulation during Hypoglycemia with Raised Circulating Nonglucose Lipid Substrates: Evidence for Regional Differences in Metabolic Capacity in the Human Brain?

Reduced Counterregulation during Hypoglycemia with Raised Circulating Nonglucose Lipid Substrates: Evidence for Regional Differences in Metabolic Capacity in the Human Brain?

Low metformin causes a more oxidized mitochondrial NADH/NAD redox state in hepatocytes and inhibits gluconeogenesis by a redox-independent mechanism

Effects of gemfibrozil on insulin resistance to fat metabolism in subjects with type 2 diabetes and hypertriglyceridaemia

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