--- module: 087-03 language: en chapter: 87 title: "Biomolecules, Protein Structure, Enzymes, Cofactors, and Biochemical Regulation" module_title: "Cofactors, biochemical regulation, pathway control, and molecular measurement" source_sha256: 35b2e75c47aeb46e7e96f1b26e26e3e368717ba777da5d46f0711afab2198e9d --- # Cofactors, regulation, pathway control, and measurement ## Vitamin-derived cofactors ### Deficiency impairs enzymes sharing a task ### Excess adds little once enzymes saturate #### Stored fat-soluble vitamins can be toxic ### Status depends on absorption through clearance ## B vitamins in energy metabolism ### Thiamine pyrophosphate #### Oxidative decarboxylation, transketolase #### Brain and heart; lactic acidosis ### Riboflavin forms flavins #### Yellow urine mainly reflects excretion ### Niacin forms NAD and NADP #### NAD catabolic, NADP biosynthetic #### Skin, gut, nervous system ### Pantothenate: coenzyme A and thioesters ### Pyridoxal phosphate and amino-group transfer #### Neuropathy, seizures, sideroblastic anaemia ## Carriers of carbon and one-carbon units ### Biotin carries activated carbon dioxide #### Avidin binding, immunoassay interference ### Folate carries one-carbon units ### B12: methionine synthase and mutase #### Folate trap causes megaloblastic change #### Folate fixes anaemia, not nerve damage ## Other vitamins and metals ### Vitamin C: hydroxylases and non-haem iron ### Vitamin K: gamma-carboxylation, warfarin ### Vitamins A and D act via nuclear receptors ### Magnesium, zinc, iron, copper, selenium, others ### Free transition metals drive harmful redox ## Mechanisms of regulation ### Substrate availability sets flux #### Saturating in-vitro assays can miss defects ### Allosteric effectors report cell state #### Feedback inhibition of committed step #### Feed-forward activation #### Reciprocal regulation prevents waste ### Covalent modification by kinases, phosphatases #### Phosphorylation has no universal meaning ### Proteolysis: activation and removal #### Synthesis and degradation set abundance ## Compartments, hormones, and control ### Compartments separate competing pathways #### Cytosolic and mitochondrial NAD pools ### Insulin stores; glucagon, catecholamines mobilise ### Longer-term hormones change capacity ### Metabolic control is distributed #### Slowest step not automatically rate-limiting #### Control shifts with feeding, exercise, disease ### Energy charge and redox ratios #### Hard to infer from plasma ## Assay principles ### Direct measurement or coupled inference ### Spectrophotometry and Beer-Lambert relation ### Mass spectrometry is highly specific ### Immunoassays: competitive or sandwich #### Cross-reaction, heterophiles, hook, biotin #### Precise yet systematically wrong ### Accuracy versus precision ### Detection limits versus clinical sensitivity ### Calibration and traceability ## Interpreting results ### Pre-analytical variation exceeds instrument error #### Haemolysis raises potassium, lactate dehydrogenase #### Ongoing metabolism consumes glucose ### Reference intervals are not treatment thresholds #### Healthy people fall outside by chance ### Dynamic tests probe regulation #### Normal baseline, abnormal response ### Omics reveal patterns and hypotheses #### Multiple testing, batch effects, validation ### Specify reaction, compartment, time, assay