Veterinary · Pre-clinical and foundation · also General Veterinary Biochemistry, Intermediary Metabolism, MSVE VPB-112, MSVE VPB-122
Biomolecules, enzymes and the metabolic pathways that turn feed into energy, milk, meat and eggs, including the ruminant's special dependence on volatile fatty acids and gluconeogenesis.
In the exams: ICAR AICE-JRF core group 08; ICAR AIEEA-PG 12 (Veterinary Biochemistry); China basic paper 12
Veterinary biochemistry is the chemistry of the living animal: the molecules a body is built from, the enzymes that change them, and the pathways that turn feed into heat, work, milk, meat, wool and eggs. It is taught in the first year of every veterinary degree, from Ludhiana to Davis to Utrecht, because every later subject leans on it. A clinician who cannot reason from a pathway cannot explain why a cobalt-deficient lamb wastes, why a fat cow goes down with ketosis a week after calving, or why a serum ALT result means something in a dog and very little in a horse.
The veterinary version differs from the human version in one large way and many small ones. The large one is the ruminant. A cow absorbs almost no glucose from her gut; she lives on volatile fatty acids made by rumen microbes and must make nearly all her glucose in the liver, mostly from propionate. Ketosis, fatty liver, pregnancy toxaemia and much of dairy herd health follow from that single fact. The small ones are species quirks with clinical weight: cats cannot make enough taurine, arginine, niacin from tryptophan, or vitamin A from carotene; birds excrete nitrogen as uric acid; guinea pigs cannot make vitamin C; hepatic enzyme patterns differ between dogs and horses.
This manual follows the twelve units of the curriculum, from biomolecules and enzymes, through carbohydrate, lipid, protein and nucleic acid metabolism, vitamins, minerals, hormones and cell signalling, to ruminant metabolism and laboratory techniques. Numbers are worked where examiners set calculations: Michaelis-Menten, Henderson-Hasselbalch, ATP yield and enzyme activity units. Clinical reference intervals are not given as numbers, because they differ by species, age, method and laboratory; use the interval printed by the laboratory that ran the test.
Water is a polar molecule, so it dissolves ions and polar compounds and forces non-polar ones together, the hydrophobic effect that drives membrane formation and protein folding. Biological structure depends on weak, non-covalent interactions: hydrogen bonds (between a hydrogen on N or O and another electronegative atom), ionic interactions (salt bridges), van der Waals forces and hydrophobic interactions. Each is weak alone, but together they hold DNA, folded proteins and antibody-antigen complexes, and because they break and re-form, structures stay dynamic.
pH is the negative log10 of the hydrogen-ion concentration. A weak acid HA dissociates to H+ and A-, and its pKa is the pH at which the two forms are equal. The Henderson-Hasselbalch equation, pH = pKa + log10([A-]/[HA]), links the three. A buffer is a weak acid with its conjugate base; it resists pH change best within about one pH unit of its pKa. The main extracellular buffer is bicarbonate: pH = 6.1 + log10([HCO3-] / (0.03 x pCO2)), with bicarbonate in mmol/L and pCO2 in mmHg. With bicarbonate 24 mmol/L and pCO2 40 mmHg, the ratio is 20 and pH is 7.40. Although its pKa of 6.1 is far from 7.4, the system is powerful because the lungs control CO2 and the kidneys control bicarbonate. Phosphate (pKa about 6.8) buffers inside cells and in urine, and proteins, especially haemoglobin with its histidine residues, buffer in blood.
Carbohydrates are polyhydroxy aldehydes or ketones and their polymers. Monosaccharides include the trioses (glyceraldehyde, dihydroxyacetone), pentoses (ribose, deoxyribose, xylose) and hexoses (glucose, galactose, mannose, fructose). Glucose in solution exists mostly as a six-membered ring in alpha and beta anomers that interconvert (mutarotation). Disaccharides join two monosaccharides by a glycosidic bond: sucrose (glucose alpha-1,2 fructose, non-reducing), lactose (galactose beta-1,4 glucose, the milk sugar) and maltose (glucose alpha-1,4 glucose).
Polysaccharides matter enormously in veterinary science because of the difference between alpha and beta bonds. Starch (amylose, linear alpha-1,4; amylopectin, alpha-1,4 with alpha-1,6 branches) and glycogen (like amylopectin but more branched) are digested by the animal's own amylases. Cellulose is a linear beta-1,4 glucose polymer; no vertebrate makes a cellulase, so only animals that host fermenting microbes, in the rumen, caecum or colon, gain energy from it. Hemicellulose and pectin are also fermented by microbes. Glycosaminoglycans such as hyaluronic acid, chondroitin sulphate and heparin are repeating disaccharide polymers found in cartilage, synovial fluid and mast cells.
Lipids are defined by insolubility in water. Fatty acids are carboxylic acids with hydrocarbon chains, saturated (palmitic C16:0, stearic C18:0) or unsaturated (oleic C18:1, linoleic C18:2 n-6, alpha-linolenic C18:3 n-3). Mammals cannot insert double bonds beyond carbon 9 counted from the carboxyl end, so linoleic and alpha-linolenic acids are essential in the diet; cats also need preformed arachidonic acid because their delta-6 desaturase activity is low. Triacylglycerols, three fatty acids esterified to glycerol, are the storage form of energy. Phospholipids (glycerophospholipids such as phosphatidylcholine, and sphingomyelin) have a polar head and two non-polar tails and form the bilayer of every membrane. Sterols, chiefly cholesterol in animals, stiffen membranes and are the precursor of bile acids, steroid hormones and vitamin D.
Proteins are chains of the twenty standard L-alpha-amino acids joined by peptide bonds. Each amino acid has an amino group, a carboxyl group and a side chain that is non-polar, polar uncharged, acidic or basic. At its isoelectric point (pI) an amino acid or protein carries no net charge; for a simple amino acid with two ionisable groups, pI is the average of the two pKa values. Protein structure is described at four levels: primary (the sequence), secondary (alpha-helix and beta-sheet held by backbone hydrogen bonds), tertiary (the overall fold, stabilised by hydrophobic interactions, hydrogen bonds, salt bridges and disulphide bonds) and quaternary (the assembly of subunits, as in haemoglobin's two alpha and two beta chains). Heat, extremes of pH, detergents and heavy metals denature proteins by disrupting these weak interactions.
Enzymes are biological catalysts, nearly all proteins. They speed reactions by lowering the activation energy, without changing the equilibrium constant or the free-energy change of the reaction. They bind substrate at an active site whose shape and chemistry favour the transition state, and binding often changes the enzyme's shape (induced fit). Many need a cofactor: a metal ion (zinc in carbonic anhydrase, magnesium with ATP) or an organic coenzyme, usually derived from a B vitamin. A tightly bound coenzyme is a prosthetic group; the protein without it is the apoenzyme, and with it the holoenzyme.
The IUBMB classifies enzymes by the reaction they catalyse and gives each an EC number. The classic six classes are oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), lyases (EC 4), isomerases (EC 5) and ligases (EC 6); a seventh class, translocases (EC 7), was added in 2018 for enzymes that move ions or molecules across membranes. Isoenzymes are different proteins that catalyse the same reaction, often in different tissues. Lactate dehydrogenase is a tetramer of H and M subunits giving five isoenzymes; creatine kinase is a dimer of M and B subunits giving CK-MM (skeletal muscle), CK-MB and CK-BB. The ratio of isoenzymes in serum can point to the tissue of origin.
For a single-substrate enzyme at steady state, initial velocity v = Vmax[S] / (Km + [S]). Vmax is the rate when the enzyme is saturated and is proportional to enzyme concentration. Km, the Michaelis constant, is the substrate concentration at which v is half of Vmax; it has units of concentration and, under simplifying assumptions, a low Km means high apparent affinity. The turnover number kcat equals Vmax divided by the molar amount of enzyme, the number of substrate molecules one active site converts per second. kcat/Km is the specificity constant.
Worked examples. If Vmax is 100 units and Km is 2 mM, then at [S] = 2 mM, v = 100 x 2 / 4 = 50; at [S] = 6 mM, v = 600 / 8 = 75; at [S] = 0.5 mM, v = 50 / 2.5 = 20. To reach 90 percent of Vmax, 0.9 = [S] / (Km + [S]), so [S] = 9 Km. This is why enzyme activity assays in the laboratory use substrate at many times Km: the rate then depends only on the amount of enzyme.
The Lineweaver-Burk double-reciprocal plot graphs 1/v against 1/[S]. It gives a straight line with y-intercept 1/Vmax, x-intercept -1/Km and slope Km/Vmax. If the y-intercept is 0.02 min per micromole, Vmax is 50 micromol/min; if the x-intercept is -0.5 per mM, Km is 2 mM.
A competitive inhibitor resembles the substrate and binds the active site; it raises apparent Km by the factor (1 + [I]/Ki) and leaves Vmax unchanged, so enough substrate overcomes it. On a Lineweaver-Burk plot the lines meet on the y-axis. Examples include malonate on succinate dehydrogenase, and ethanol competing with ethylene glycol for alcohol dehydrogenase, the basis of treating antifreeze poisoning in dogs. A pure non-competitive inhibitor binds equally to enzyme and enzyme-substrate complex, lowers Vmax by the same factor and leaves Km unchanged; lines meet on the x-axis. An uncompetitive inhibitor binds only the enzyme-substrate complex and lowers both Vmax and Km, giving parallel lines. Irreversible inhibitors, such as organophosphates that phosphorylate the serine of acetylcholinesterase, destroy activity until new enzyme is made.
Cells regulate enzymes by allostery (effectors at sites other than the active site, giving sigmoid kinetics, as ATP and AMP do on phosphofructokinase-1), by covalent modification (phosphorylation of glycogen phosphorylase and glycogen synthase), by proteolytic activation of zymogens (trypsinogen to trypsin; clotting factors), and by changing the amount of enzyme through gene expression.
Enzyme activity is expressed as the international unit, U: the amount of enzyme that converts one micromole of substrate per minute under defined conditions. The SI unit is the katal, one mole per second; 1 U = 1/60 micromol/s = 16.67 nkat. Specific activity is units per milligram of protein and rises as an enzyme is purified; the fold purification is the ratio of specific activities. Because activity depends on temperature, pH and substrate, a result in U/L is meaningful only with the method and temperature, which is one reason reference intervals are laboratory-specific.
Serum enzymes leak from damaged cells or are induced. Their value depends on tissue specificity, which differs by species. Alanine aminotransferase (ALT) is concentrated in hepatocyte cytoplasm in dogs and cats, so it is a sensitive marker of hepatocellular injury there; in horses, cattle, sheep and pigs hepatic ALT activity is low and it is of little use. In large animals sorbitol dehydrogenase (SDH) and glutamate dehydrogenase (GLDH) serve as liver-specific leakage enzymes. Aspartate aminotransferase (AST) is present in liver and muscle, so a rise must be read alongside creatine kinase (CK): CK is muscle-specific and rises and falls quickly, while AST stays high longer, so high AST with normal CK suggests liver or an older muscle insult.
Gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) are induced enzymes that rise with cholestasis. GGT is the more useful of the two in ruminants and horses, where ALP varies widely. In the dog, glucocorticoids induce a distinct ALP isoenzyme, so high ALP in a dog may reflect endogenous or given steroids rather than liver disease; the cat has low hepatic ALP with a short half-life, so even a modest rise in a cat is significant. Young growing animals have high bone ALP. Colostrum-fed neonatal calves and lambs have high serum GGT absorbed from colostrum, which is used as a rough indicator of passive transfer. Amylase and lipase have been used for pancreatitis, but species-specific pancreatic lipase immunoassays are now preferred in dogs and cats. Every interpretation must use the interval from the laboratory and species concerned.
Glycolysis, in the cytosol of every cell, splits one glucose into two pyruvate, with a net gain of 2 ATP and 2 NADH. Three steps are essentially irreversible and are the control points: hexokinase (or glucokinase in liver), phosphofructokinase-1 (PFK-1) and pyruvate kinase. PFK-1 is the main pace-setter; it is activated by AMP and fructose-2,6-bisphosphate and inhibited by ATP and citrate. Glucokinase has a high Km for glucose and is not inhibited by its product, which suits the liver's role of taking up glucose only when blood glucose is high. The cat's liver has very little glucokinase activity, one reason cats handle a large glucose load poorly.
Pyruvate has several fates. Without enough oxygen, as in sprinting muscle or red cells that lack mitochondria, lactate dehydrogenase reduces it to lactate to regenerate NAD+; glucose to lactate yields 2 ATP per glucose, and glycogen to lactate yields 3 ATP per glucosyl unit because phosphorolysis saves the hexokinase ATP. Lactate returns to the liver for gluconeogenesis, the Cori cycle. In mitochondria, the pyruvate dehydrogenase complex oxidatively decarboxylates pyruvate to acetyl-CoA; it needs thiamine pyrophosphate, lipoic acid, coenzyme A, FAD and NAD+, and it is irreversible, so acetyl-CoA cannot be turned back into glucose. Pyruvate carboxylase, which needs biotin and is activated by acetyl-CoA, makes oxaloacetate, the first step of gluconeogenesis and the way the TCA cycle is refilled.
The tricarboxylic acid (citric acid, Krebs) cycle in the mitochondrial matrix condenses acetyl-CoA with oxaloacetate to citrate and oxidises the two carbons to CO2. Each turn gives 3 NADH, 1 FADH2 and 1 GTP (or ATP). The electron transport chain passes electrons from NADH (complex I) and FADH2 (complex II) through ubiquinone, complex III, cytochrome c and complex IV to oxygen, pumping protons out of the matrix; ATP synthase uses the proton gradient to make ATP. Using the modern P/O ratios of about 2.5 for NADH and 1.5 for FADH2, one acetyl-CoA yields 10 ATP. Older texts use 3 and 2 and quote 12; examiners accept whichever basis is stated, so always state it.
For one glucose oxidised completely, glycolysis gives 2 ATP and 2 cytosolic NADH, pyruvate dehydrogenase gives 2 NADH, and two turns of the cycle give 6 NADH, 2 FADH2 and 2 GTP. The total is 30 or 32 ATP depending on whether cytosolic NADH enters mitochondria by the glycerol-3-phosphate shuttle (1.5 each) or the malate-aspartate shuttle (2.5 each); older texts give 36 or 38. Uncouplers such as 2,4-dinitrophenol, and the brown-fat protein thermogenin (UCP1) in newborn lambs, calves and hibernators, let protons back without making ATP and release the energy as heat. Cyanide blocks complex IV, which is why cyanogenic plants such as sorghum regrowth kill livestock quickly, and fluoroacetate is converted to fluorocitrate, which blocks aconitase.
Glycogen is stored mainly in liver, which releases glucose to the blood, and in muscle, which uses its glycogen itself because it lacks glucose-6-phosphatase. Glycogen synthase adds UDP-glucose units by alpha-1,4 bonds and the branching enzyme makes alpha-1,6 branches; glycogen phosphorylase, with pyridoxal phosphate, releases glucose-1-phosphate. Glucagon and adrenaline, acting through cyclic AMP and protein kinase A, activate phosphorylase and inactivate synthase; insulin does the reverse. Inherited glycogen disorders occur in animals: glycogen branching enzyme deficiency in Quarter Horse lineages, and polysaccharide storage myopathy type 1, linked to a glycogen synthase (GYS1) mutation, in several horse breeds.
The pentose phosphate pathway oxidises glucose-6-phosphate to make NADPH and ribose-5-phosphate. NADPH is needed for fatty acid and steroid synthesis and to keep glutathione reduced in red cells. Its first enzyme is glucose-6-phosphate dehydrogenase; transketolase in its non-oxidative arm needs thiamine pyrophosphate. Oxidant plants, such as onion and garlic in dogs and cats and red maple leaves in horses, overwhelm this defence and cause Heinz-body haemolytic anaemia.
Gluconeogenesis makes glucose from non-carbohydrate precursors, mainly in liver and to a lesser extent kidney cortex. It reverses glycolysis but bypasses the three irreversible steps: pyruvate carboxylase and phosphoenolpyruvate carboxykinase (PEPCK) bypass pyruvate kinase, fructose-1,6-bisphosphatase bypasses PFK-1, and glucose-6-phosphatase bypasses hexokinase. Making one glucose from two pyruvate costs 4 ATP, 2 GTP and 2 NADH. Substrates are lactate, glycerol, glucogenic amino acids and, in ruminants, propionate. Even-chain fatty acids cannot give net glucose, because acetyl-CoA carbons are lost as CO2 in the cycle. Cats, as obligate carnivores, keep hepatic gluconeogenesis from amino acids running even in the fed state.
Dietary triacylglycerol is emulsified by bile salts, hydrolysed by pancreatic lipase, absorbed and re-esterified in enterocytes, then exported in chylomicrons into lymph. The liver exports triacylglycerol in very-low-density lipoprotein (VLDL). Lipoprotein lipase on capillary endothelium hydrolyses the triacylglycerol of both so that tissues can take up fatty acids; the remnants return to the liver. Low-density lipoprotein (LDL) carries cholesterol to tissues and high-density lipoprotein (HDL) carries it back. Species differ: dogs, cats, horses and cattle carry most of their plasma cholesterol in HDL and are relatively resistant to atherosclerosis, whereas in humans and pigs LDL predominates.
In negative energy balance, hormone-sensitive lipase and adipose triglyceride lipase in adipose tissue release non-esterified fatty acids (NEFA) and glycerol. Adrenaline, noradrenaline and glucagon stimulate this through cyclic AMP; insulin inhibits it strongly. NEFA travel on albumin. Glycerol goes to the liver for gluconeogenesis. Serum NEFA concentration is therefore a direct index of fat mobilisation and is used in transition dairy cows.
Fatty acids are activated in the cytosol to acyl-CoA by acyl-CoA synthetase, which splits ATP to AMP, costing two high-energy phosphate bonds. Long-chain acyl groups enter the mitochondrion on carnitine: carnitine palmitoyltransferase I (CPT-I) on the outer membrane is the rate-limiting step and is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, so synthesis and oxidation do not run together. Each cycle of beta-oxidation removes two carbons as acetyl-CoA and produces one FADH2 and one NADH.
Palmitate (C16) needs 7 cycles and gives 8 acetyl-CoA, 7 FADH2 and 7 NADH. With 10 ATP per acetyl-CoA, 1.5 per FADH2 and 2.5 per NADH: 80 + 10.5 + 17.5 = 108, minus 2 for activation, equals 106 ATP. Stearate (C18) gives 9 acetyl-CoA from 8 cycles: 90 + 12 + 20 - 2 = 120 ATP. Older texts, using P/O values of 3 and 2 and 12 ATP per acetyl-CoA, give 129 for palmitate. Odd-chain fatty acids leave one propionyl-CoA, which is glucogenic by the route described for ruminants.
When fatty acids flood the liver and oxaloacetate is scarce, acetyl-CoA is diverted into ketone bodies in liver mitochondria. Two acetyl-CoA form acetoacetyl-CoA; HMG-CoA synthase, the regulated step, adds a third to make 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which HMG-CoA lyase splits to acetoacetate. Acetoacetate is reduced to beta-hydroxybutyrate (BHB) or decarboxylates spontaneously to acetone, which gives the sweet breath smell of ketotic cows. The liver cannot use ketone bodies because it lacks succinyl-CoA:3-oxoacid CoA transferase; heart, muscle, kidney and, after adaptation, brain oxidise them. Oxidising one BHB yields 1 NADH at BHB dehydrogenase (2.5 ATP) and two acetyl-CoA (20 ATP), minus the succinyl-CoA used by the transferase (1 GTP equivalent): 21.5 ATP.
De novo fatty acid synthesis occurs in the cytosol. Acetyl-CoA carboxylase, a biotin enzyme and the committed step, makes malonyl-CoA; it is activated by citrate and insulin and inhibited by long-chain acyl-CoA, glucagon and AMP-activated protein kinase. Fatty acid synthase then adds two carbons at a time using NADPH, stopping at palmitate. The site and carbon source of lipogenesis differ by species. In ruminants adipose tissue is the main site and acetate the main carbon source; their adipose tissue has low ATP-citrate lyase activity, so glucose carbon is spared for other uses, and NADPH comes partly from the pentose phosphate pathway and partly from NADP-isocitrate dehydrogenase. In pigs, too, adipose tissue dominates, while in birds the liver makes most fatty acid. The mammary gland of ruminants makes the short- and medium-chain fatty acids of milk fat from acetate and BHB.
Cholesterol is synthesised from acetyl-CoA via HMG-CoA; cytosolic HMG-CoA reductase is the rate-limiting enzyme and the target of statins. Cholesterol is converted to bile acids in the liver. Arachidonic acid released from phospholipids by phospholipase A2 is converted by cyclo-oxygenases to prostaglandins and thromboxanes, the target of non-steroidal anti-inflammatory drugs, and by lipoxygenases to leukotrienes.
Body proteins turn over continuously; amino acids beyond immediate need are not stored but catabolised. The first step is usually transamination: an aminotransferase, with pyridoxal phosphate from vitamin B6, moves the amino group to alpha-ketoglutarate to make glutamate. ALT transfers between alanine and pyruvate, AST between aspartate and oxaloacetate. Glutamate dehydrogenase then releases the nitrogen as ammonia in the liver mitochondria. Ammonia is toxic to the brain, and muscle and other tissues send nitrogen to the liver safely as glutamine and alanine.
In mammals the urea cycle in periportal hepatocytes turns ammonia into urea. Carbamoyl phosphate synthetase I, activated by N-acetylglutamate, combines ammonia, bicarbonate and 2 ATP to make carbamoyl phosphate in the mitochondrion; ornithine transcarbamoylase adds it to ornithine to make citrulline; in the cytosol argininosuccinate synthetase adds aspartate, argininosuccinate lyase releases fumarate and arginine, and arginase cleaves arginine to urea and ornithine. One urea costs 3 ATP (4 high-energy bonds) and carries one nitrogen from ammonia and one from aspartate. Cats cannot make enough arginine or ornithine for the cycle; a single arginine-free meal can cause hyperammonaemia within hours. Birds are uricotelic: they lack a functional hepatic urea cycle, excrete nitrogen as uric acid, and need arginine in the diet. Liver failure or a portosystemic shunt raises blood ammonia and lowers blood urea, causing hepatic encephalopathy.
After removal of nitrogen, the carbon skeletons enter central metabolism. Glucogenic amino acids give pyruvate, oxaloacetate, alpha-ketoglutarate, succinyl-CoA or fumarate and can make glucose; this is a major glucose source in fasting, in carnivores and in ruminants. Leucine and lysine are purely ketogenic, giving acetyl-CoA or acetoacetate. Isoleucine, phenylalanine, tyrosine, tryptophan and threonine are both. Essential amino acids for most mammals are those the body cannot make fast enough: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, with arginine essential for cats, birds and young growing animals. Methionine and lysine are commonly the first-limiting amino acids in cereal-based pig, poultry and dairy diets.
Amino acids are precursors of many small molecules. Tyrosine gives catecholamines, thyroid hormones and melanin; tryptophan gives serotonin, melatonin and, except effectively in cats, niacin; glycine and succinyl-CoA give haem, the first step being catalysed by delta-aminolaevulinate synthase, while lead inhibits later steps (ALA dehydratase and ferrochelatase); arginine gives nitric oxide and, with glycine and methionine, creatine; histidine gives histamine; glutamate gives GABA. Cats need dietary taurine because their synthesis from cysteine is limited and they conjugate bile acids only with taurine; deficiency causes central retinal degeneration and dilated cardiomyopathy. Glutathione, a tripeptide of glutamate, cysteine and glycine, is the main intracellular antioxidant.
Ruminants add a microbial layer. Rumen microbes degrade much of dietary protein to peptides, amino acids and ammonia, and use ammonia plus fermentable energy to make microbial protein, which then flows to the abomasum and small intestine and supplies much of the cow's absorbed amino acids. Non-protein nitrogen, such as urea, can therefore replace part of the protein in the ration, provided enough fermentable carbohydrate is present to capture the ammonia. Urea is also recycled: blood urea diffuses into saliva and across the rumen wall, where microbial urease converts it to ammonia, a nitrogen-saving loop important on low-protein forage.
If ammonia is released faster than microbes can use it, as when urea is fed carelessly or animals break into urea stores, rumen pH rises, more ammonia is in the un-ionised form that crosses membranes, the liver's urea capacity is exceeded, and the animal shows ammonia toxicity: tremors, bloat and death. Blood and milk urea concentrations are used in dairy herds to judge the balance of rumen-degradable protein and energy; interpretation thresholds differ between laboratories and countries.
A nucleotide has a nitrogenous base, a pentose sugar and one or more phosphate groups; without phosphate it is a nucleoside. The purines are adenine and guanine; the pyrimidines are cytosine, thymine (DNA) and uracil (RNA). DNA uses 2-deoxyribose and RNA uses ribose. Nucleotides link through 3'-5' phosphodiester bonds, giving a strand with a 5' and a 3' end. DNA is usually a right-handed double helix (B-form) of two antiparallel strands, with adenine pairing thymine by two hydrogen bonds and guanine pairing cytosine by three. Chargaff's rule follows: in double-stranded DNA, A equals T and G equals C, so if A is 20 percent, T is 20, and G and C are 30 each. G-C-rich DNA melts at a higher temperature. Heating separates the strands (denaturation), raising absorbance at 260 nm, the hyperchromic effect.
In eukaryotic nuclei DNA wraps around histone octamers to form nucleosomes and chromatin. RNA is usually single-stranded and comes in several kinds: messenger RNA carries the code, transfer RNA carries amino acids with an anticodon, ribosomal RNA forms the catalytic core of the ribosome, and small regulatory RNAs such as microRNAs silence genes. Nucleotides also serve as energy currency (ATP, GTP), second messengers (cyclic AMP, cyclic GMP) and parts of coenzymes (NAD, FAD, coenzyme A).
DNA replication is semi-conservative: each daughter duplex has one old and one new strand. DNA polymerases synthesise only 5' to 3' and need a primer, so the leading strand is continuous and the lagging strand is made in Okazaki fragments joined by DNA ligase. Proofreading and repair systems keep errors extremely low. Transcription by RNA polymerase copies a gene into RNA; in eukaryotes the primary transcript is capped at the 5' end, polyadenylated at the 3' end and spliced to remove introns. Translation on ribosomes reads codons of three bases; the genetic code has 64 codons for 20 amino acids plus stop signals, is degenerate and is nearly universal. AUG codes for methionine and starts translation. Many antibacterial drugs target the bacterial 70S ribosome (tetracyclines, aminoglycosides, macrolides, chloramphenicol), which differs from the eukaryotic 80S ribosome.
Purine rings are built on ribose-5-phosphate from glycine, glutamine, aspartate, CO2 and one-carbon units from folate, reaching IMP, which branches to AMP and GMP. Pyrimidines are made as a ring first (from carbamoyl phosphate made by cytosolic carbamoyl phosphate synthetase II, and aspartate) and then attached to ribose. Ribonucleotide reductase makes deoxyribonucleotides, and thymidylate synthase, using a folate cofactor, makes dTMP; this is why folate antagonists and B12 deficiency impair cell division. Salvage pathways, notably hypoxanthine-guanine phosphoribosyltransferase, recycle bases.
Purines are degraded to hypoxanthine, xanthine and uric acid by xanthine oxidase, a molybdenum enzyme inhibited by allopurinol. Most mammals then oxidise uric acid to more soluble allantoin with uricase. Humans and apes lack uricase. Dalmatian dogs have a defect in a urate transporter (SLC2A9) in liver and kidney, so they excrete much uric acid and are prone to urate uroliths; dogs with portosystemic shunts also form urate stones. Birds and reptiles excrete uric acid as their main nitrogen end-product, and gout occurs in poultry when renal excretion fails. In ruminants, urinary excretion of purine derivatives (allantoin, uric acid, xanthine and hypoxanthine) comes largely from microbial nucleic acids digested in the intestine, and is used in research as an index of microbial protein supply.
Most water-soluble vitamins work as coenzymes. Thiamine (B1) becomes thiamine pyrophosphate, needed by pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched-chain keto-acid dehydrogenase and transketolase. Deficiency starves the brain of energy. In ruminants, polioencephalomalacia (cerebrocortical necrosis) is linked to thiamine deficiency from rumen thiaminases and to high dietary sulphur; horses eating bracken fern and carnivores fed raw fish containing thiaminase develop thiamine deficiency. Riboflavin (B2) forms FMN and FAD for flavoprotein oxidoreductases. Niacin (B3) forms NAD+ and NADP+. Most mammals can make niacin from tryptophan, but cats cannot make enough because of high picolinic carboxylase activity, so niacin is required in feline diets. Pantothenic acid (B5) is part of coenzyme A. Pyridoxine (B6) becomes pyridoxal phosphate for aminotransferases, decarboxylases, glycogen phosphorylase and ALA synthase.
Biotin (B7) is the coenzyme of carboxylases: pyruvate carboxylase, acetyl-CoA carboxylase and propionyl-CoA carboxylase. Raw egg white contains avidin, which binds biotin. Folate carries one-carbon units as tetrahydrofolate for purine and thymidylate synthesis. Cobalamin (B12) is needed by methylmalonyl-CoA mutase and methionine synthase. Rumen microbes make B12 only if the diet supplies cobalt, and ruminants need a great deal of it to convert propionate to succinyl-CoA; cobalt deficiency therefore causes wasting and ill-thrift in sheep and cattle on cobalt-poor soils. In dogs and cats, low serum cobalamin is common in chronic intestinal disease and exocrine pancreatic insufficiency.
Ascorbic acid is a reducing agent and the cofactor of prolyl and lysyl hydroxylases in collagen synthesis. Most domestic animals synthesise it from glucose in liver (or kidney in some birds and reptiles), but guinea pigs, primates and some fish and bats lack L-gulonolactone oxidase and need it in the diet; guinea pigs on stale feed develop scurvy.
Vitamin A (retinol) is formed from beta-carotene by intestinal cleavage in most species. Cats lack effective cleavage and need preformed vitamin A. 11-cis-retinal combines with opsin to form rhodopsin in rod cells; retinoic acid acts through nuclear receptors on epithelial differentiation. Deficiency causes night blindness, squamous metaplasia and reproductive failure; excess causes bone lesions, seen in cats fed mostly liver. Vitamin D3 (cholecalciferol) forms in skin from 7-dehydrocholesterol under ultraviolet light, but dogs and cats make little and rely on the diet; D2 comes from plants. Liver 25-hydroxylation and kidney 1-alpha-hydroxylation, stimulated by parathyroid hormone and low phosphate, make calcitriol, which raises intestinal calcium and phosphate absorption. Vitamin E (alpha-tocopherol) protects membrane lipids from peroxidation and works with selenium-dependent glutathione peroxidase; combined deficiency causes nutritional myodegeneration (white muscle disease) in calves and lambs and mulberry heart disease in pigs. Vitamin K is the cofactor of gamma-glutamyl carboxylase, which makes clotting factors II, VII, IX and X able to bind calcium. Coumarin rodenticides and dicoumarol from mouldy sweet clover block vitamin K epoxide reductase, causing bleeding.
Calcium is the most abundant mineral in the body, nearly all in bone as hydroxyapatite. The small ionised fraction in plasma controls nerve and muscle excitability, clotting and hormone secretion, and acts as a second messenger. About half of plasma calcium is ionised, most of the rest bound to albumin, so total calcium falls with hypoalbuminaemia while ionised calcium may be normal; acidosis increases the ionised fraction. Parathyroid hormone, calcitriol and calcitonin regulate it. Parturient paresis (milk fever) in dairy cows follows the sudden calcium drain of colostrum and milk; diets with a negative dietary cation-anion difference before calving improve the response of bone and kidney to parathyroid hormone. Phosphorus is part of ATP, nucleic acids, phospholipids and bone, and phosphate buffers urine. Magnesium is the cofactor of every reaction that uses ATP, which is carried as Mg-ATP; low magnesium on lush, potassium-rich spring pasture causes grass tetany (hypomagnesaemic tetany) in cattle and sheep, because the ruminant has no large mobilisable magnesium store and absorption from the rumen is reduced by high potassium.
Sodium, potassium and chloride set osmolality, membrane potential and acid-base balance. Plasma osmolality can be estimated as 2 x (Na + K) + glucose + urea, all in mmol/L; a large gap between measured and calculated values suggests an unmeasured osmole such as ethylene glycol. The anion gap, (Na + K) - (Cl + HCO3), estimates unmeasured anions; it rises with lactate, ketoacids, uraemic acids and glycolate from ethylene glycol.
Most trace elements work as parts of enzymes or proteins. Iron is in haem (haemoglobin, myoglobin, cytochromes) and in iron-sulphur proteins; it is carried by transferrin and stored as ferritin and haemosiderin. Piglets are born with small iron stores and sow milk is low in iron, so indoor-reared piglets become anaemic without supplementation. Copper is part of cytochrome c oxidase, lysyl oxidase (collagen and elastin cross-linking), tyrosinase (melanin), superoxide dismutase and ceruloplasmin. Deficiency causes enzootic ataxia (swayback) in lambs, loss of coat colour, bone fragility and anaemia. In the rumen, molybdenum and sulphur form thiomolybdates that bind copper, so excess molybdenum or sulphur causes secondary copper deficiency. Sheep store copper readily in the liver and excrete it poorly in bile, so they are unusually prone to chronic copper poisoning with a haemolytic crisis; Bedlington Terriers have an inherited copper storage hepatopathy.
Zinc is in over 300 enzymes, including carbonic anhydrase, alkaline phosphatase, alcohol dehydrogenase and superoxide dismutase, and in zinc-finger transcription factors; deficiency causes parakeratosis in pigs. Selenium is incorporated as selenocysteine into glutathione peroxidases and the iodothyronine deiodinases that convert T4 to T3. Iodine is part of thyroxine and triiodothyronine; deficiency causes goitre and weak or hairless newborns. Manganese is a cofactor of pyruvate carboxylase, arginase and mitochondrial superoxide dismutase, and deficiency causes perosis in chicks. Molybdenum is in xanthine oxidase, sulphite oxidase and aldehyde oxidase. Cobalt's only known role in animals is as part of vitamin B12. Soil levels of copper, cobalt, selenium and iodine vary widely by region, which is why deficiency diseases are local.
Hormones are chemical messengers carried in blood to distant target cells. By chemistry they are peptides and proteins (insulin, glucagon, growth hormone, prolactin, the gonadotrophins, parathyroid hormone), amino acid derivatives (catecholamines and thyroid hormones from tyrosine, melatonin from tryptophan), steroids (cortisol, aldosterone, oestradiol, progesterone, testosterone, and calcitriol, strictly a secosteroid) and eicosanoids (prostaglandin F2-alpha, which lyses the corpus luteum in cattle and is used to synchronise oestrus). Water-soluble hormones, peptides and catecholamines, cannot cross the membrane and act on cell-surface receptors that trigger second messengers; their effects are fast. Steroid and thyroid hormones are lipid-soluble, travel bound to carrier proteins, enter cells and bind intracellular receptors that act as transcription factors; their effects take hours and involve new protein synthesis.
Thyroid hormone synthesis shows biochemistry clearly: iodide is trapped by the sodium-iodide symporter, oxidised by thyroid peroxidase and attached to tyrosine residues of thyroglobulin, and coupled iodotyrosines give T4 and T3. Most circulating hormone is T4; selenium-dependent deiodinases in tissues convert it to the more active T3. Steroid hormones all derive from cholesterol; the rate-limiting step is its transfer into mitochondria by the steroidogenic acute regulatory protein and cleavage of its side chain to pregnenolone.
Insulin, from pancreatic beta cells, is the hormone of the fed state. It promotes glucose uptake in muscle and adipose tissue through GLUT4, glycogen synthesis, lipogenesis and protein synthesis, and it inhibits lipolysis, gluconeogenesis and ketogenesis. Glucagon, from alpha cells, is the hormone of the fasted state, acting mainly on the liver to stimulate glycogenolysis, gluconeogenesis and ketogenesis. Adrenaline acts quickly in stress. Cortisol promotes gluconeogenesis from amino acids and is permissive for glucagon and adrenaline. Growth hormone is anabolic for protein, largely through insulin-like growth factor 1 (IGF-1) from the liver, but antagonises insulin in fat and muscle and promotes lipolysis.
In the ruminant, propionate as well as glucose stimulates insulin secretion. Around calving the dairy cow shows homeorhesis, the coordinated redirection of nutrients towards the udder: insulin falls, peripheral tissues become less sensitive to insulin, growth hormone rises, and hepatic growth hormone receptors fall so that IGF-1 drops, an 'uncoupled' somatotropic axis. The result is that glucose is spared for lactose synthesis and fat is mobilised, which is necessary but is also the starting point of ketosis and fatty liver.
Cell-surface receptors turn an outside signal into an inside response. The largest family is the G-protein-coupled receptors, seven-transmembrane proteins linked to heterotrimeric G proteins made of alpha, beta and gamma subunits. When a ligand binds, the alpha subunit exchanges GDP for GTP and separates to act on an effector; its own GTPase activity then switches it off. Gs activates adenylyl cyclase, raising cyclic AMP, which activates protein kinase A (PKA); this is how glucagon and beta-adrenergic agonists trigger glycogenolysis and lipolysis. Gi inhibits adenylyl cyclase, as alpha-2 adrenergic agonists used for sedation do. Gq activates phospholipase C-beta, which splits phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3), releasing calcium from the endoplasmic reticulum, and diacylglycerol (DAG), which with calcium activates protein kinase C. Calcium acts through calmodulin on many enzymes, including phosphorylase kinase.
Signals are ended by phosphodiesterases that break cyclic AMP and cyclic GMP (inhibited by methylxanthines such as theophylline and theobromine, one reason chocolate is toxic to dogs), by phosphatases, and by receptor desensitisation. Bacterial toxins exploit these switches: cholera toxin and the related Escherichia coli heat-labile enterotoxin ADP-ribosylate Gs-alpha so it stays active, flooding enterocytes with cyclic AMP and causing secretory diarrhoea; pertussis toxin ADP-ribosylates Gi so it cannot inhibit. Nitric oxide diffuses into smooth muscle and activates soluble guanylyl cyclase, making cyclic GMP and relaxation.
Receptor tyrosine kinases, such as the insulin and IGF-1 receptors and many growth factor receptors, phosphorylate themselves and docking proteins on tyrosine. For insulin, the receptor phosphorylates insulin receptor substrates, which activate phosphatidylinositol 3-kinase and then protein kinase B (Akt); Akt drives GLUT4 to the membrane and switches on glycogen synthesis, while the Ras-MAP kinase branch drives growth. Cytokine receptors, including those for growth hormone, prolactin, leptin and erythropoietin, have no kinase of their own but use associated Janus kinases (JAKs) to phosphorylate STAT proteins that move to the nucleus; JAK inhibitors are used in canine allergic dermatitis.
Nuclear receptors are ligand-activated transcription factors: receptors for glucocorticoids, mineralocorticoids, sex steroids, thyroid hormone, calcitriol and retinoic acid, and the peroxisome proliferator-activated receptors (PPARs), which respond to fatty acids and control genes of fatty acid oxidation and storage. Two intracellular sensors integrate nutrient status. AMP-activated protein kinase (AMPK) senses a high AMP:ATP ratio and switches off anabolic processes such as fatty acid synthesis (by phosphorylating acetyl-CoA carboxylase) while switching on catabolism. The mechanistic target of rapamycin (mTOR) senses amino acids and growth factors and drives protein synthesis, including milk protein synthesis in the mammary gland.
In the rumen, bacteria, protozoa, fungi and archaea ferment cellulose, hemicellulose, starch and sugars to volatile fatty acids (VFA), mainly acetate (C2), propionate (C3) and butyrate (C4), with smaller amounts of branched-chain VFA from amino acids, plus CO2 and methane. Archaea make methane from CO2 and hydrogen, which removes hydrogen and keeps fermentation running but wastes a share of feed energy. VFA supply the majority of the ruminant's metabolisable energy. On forage diets acetate dominates, typically around two-thirds of the VFA, with propionate and butyrate much smaller; high-grain diets raise the proportion of propionate and lower acetate. The exact proportions vary with diet, time after feeding and method, so treat any single figure as illustrative. Ionophores fed to cattle shift fermentation towards propionate.
VFA are weak acids with pKa values near 4.8 (acetic acid about 4.76). At a normal rumen pH around 6 to 7 they are largely dissociated; at pH 6.76 the acetate-to-acetic acid ratio is 10 to the power 2, or 100:1. They are absorbed across the rumen wall, partly in the un-ionised form, and absorption brings in protons that help buffer the rumen; saliva supplies bicarbonate and phosphate buffer. In acute grain overload, rapid starch fermentation produces lactic acid, whose pKa of about 3.86 is roughly one unit lower than that of VFA; at pH 5.0 lactic acid is about 93 percent dissociated against about 63 percent for acetic acid, so lactate drives rumen pH down harder, and absorbed lactate, especially the slowly metabolised D-isomer, contributes to systemic acidosis. The rumen epithelium metabolises much of the butyrate to beta-hydroxybutyrate, so BHB is present in a healthy fed ruminant.
The three VFA go to different places. Propionate is taken up almost completely by the liver on first pass, and in the fed ruminant it is the main substrate for gluconeogenesis. Acetate largely passes the liver and is used by peripheral tissues for energy and, in adipose tissue and the mammary gland, as the main carbon source for fatty acid synthesis. Butyrate reaches the liver mostly as BHB, is used by tissues for energy and by the udder for milk fat. Neither acetate nor butyrate can produce net glucose.
The route from propionate to glucose is examined often. Propionate is activated to propionyl-CoA (using ATP). Propionyl-CoA carboxylase, a biotin enzyme, adds CO2 to make D-methylmalonyl-CoA. Methylmalonyl-CoA epimerase (racemase) converts it to L-methylmalonyl-CoA, and methylmalonyl-CoA mutase, which needs adenosylcobalamin (vitamin B12), rearranges it to succinyl-CoA. Succinyl-CoA enters the TCA cycle and is converted to oxaloacetate, which PEPCK turns into phosphoenolpyruvate for gluconeogenesis. Because two three-carbon molecules make one glucose, the theoretical maximum is one mole of glucose per two moles of propionate. In cobalt deficiency, mutase activity falls, methylmalonic acid accumulates and propionate is poorly used, so appetite falls and animals waste. The same pathway handles odd-chain fatty acids and the carbon skeletons of valine, isoleucine, methionine and threonine.
A ruminant absorbs little glucose from the intestine on most diets, because rumen microbes ferment almost all dietary sugar and starch; some starch escapes to the small intestine on high-grain diets. The liver therefore makes most of the glucose the animal needs, and hepatic gluconeogenesis continues even after feeding, the opposite of the monogastric pattern. Propionate is the largest single substrate in the fed animal; amino acids, lactate and glycerol contribute the rest, and the amino-acid share rises in underfed animals and in early lactation. Ruminant liver has very low glucokinase activity and takes up little glucose, and ruminant adipose tissue makes fatty acid from acetate rather than glucose; both spare glucose.
The dairy cow's need for glucose is dominated by the udder, which uses glucose to make lactose, the osmotic regulator of milk volume. Each lactose molecule is made from two hexoses, glucose and galactose (itself made from glucose), so each kilogram of lactose needs a little more than a kilogram of glucose. For example, a cow producing 30 kg milk containing 4.8 percent lactose secretes 1.44 kg lactose, 4.21 mol; that needs 8.41 mol glucose, about 1.52 kg, for lactose alone. Late pregnancy adds a large glucose demand from the fetus and placenta, particularly in ewes carrying twins or triplets. When supply fails to meet these demands, the result is hypoglycaemia, fat mobilisation and ketosis.
In early lactation, milk output rises faster than feed intake, so the cow is in negative energy balance. Low insulin and insulin resistance allow lipolysis, and NEFA flood into the blood. The liver takes up NEFA in proportion to their blood concentration. There they can be oxidised completely in the TCA cycle, partly oxidised to ketone bodies, or re-esterified to triacylglycerol. Three things push the balance toward ketogenesis. First, oxaloacetate is drawn off for gluconeogenesis, so less acetyl-CoA can enter the cycle. Second, low insulin and low malonyl-CoA lift the brake on CPT-I, so more fatty acid enters mitochondria. Third, high NEFA inflow simply exceeds oxidative capacity. BHB and acetoacetate rise in blood, milk and urine; acetone may be smelt on the breath. Clinical signs include falling milk yield, reduced appetite (concentrates refused first), weight loss and sometimes nervous signs. Subclinical ketosis, raised BHB without clinical signs, is common and is linked to displaced abomasum, metritis, lower milk yield and poorer fertility. Cow-side and laboratory BHB cut-points are used to define it; the cut-point differs between studies and laboratories, and blood, milk and urine tests are not interchangeable.
Textbooks describe a hypoglycaemic, low-insulin type at peak milk demand a few weeks after calving, and a type with fatty liver and insulin resistance in the first two weeks, often in cows over-conditioned at calving. Ketosis can also follow feeding silage rich in butyric acid, because butyrate becomes BHB in the rumen wall.
Fatty liver (hepatic lipidosis) develops when re-esterified triacylglycerol accumulates faster than the liver can export it as VLDL. Ruminant liver exports VLDL slowly, so any large NEFA surge around calving leaves fat in hepatocytes. Severe fatty liver impairs gluconeogenesis, urea synthesis and detoxification, and is associated with raised liver enzymes, especially AST, GLDH and SDH, and raised bilirubin and NEFA. Liver biopsy fat content is the definitive measure. Treatment aims to raise glucose and insulin and cut lipolysis: intravenous glucose, oral glucogenic precursors such as propylene glycol, which the liver converts to glucose via lactate and pyruvate, and glucocorticoids in some protocols, all at doses to be confirmed against the product label and your national formulary. Pregnancy toxaemia of ewes and does is the same biochemistry driven by fetal glucose demand in late pregnancy.
Most clinical chemistry rests on photometry. The Beer-Lambert law states that absorbance A = epsilon x c x l, where epsilon is the molar absorption coefficient, c the concentration and l the path length. Absorbance is the log of incident over transmitted light and has no units. NADH absorbs at 340 nm and NAD+ does not, with epsilon of 6.22 per mM per cm (6,220 per M per cm), so many enzyme and substrate assays are designed to produce or consume NADH and are read at 340 nm. Nucleic acids absorb at 260 nm and proteins at 280 nm; an A260/A280 ratio near 1.8 suggests reasonably pure DNA, and an A260 of 1.0 corresponds to about 50 micrograms per mL of double-stranded DNA.
Kinetic enzyme assays measure the rate of absorbance change. Activity in U/L = (delta A per min / epsilon in per mM per cm, path 1 cm) x (total volume / sample volume) x 1000. For example, a change of 0.155 per minute at 340 nm, 1.0 mL total volume and 0.05 mL serum gives (0.155 / 6.22) x 20 x 1000 = 498 U/L. If activity exceeds the linear range, the sample is diluted and the result multiplied by the dilution factor. Proteins are measured by biuret, Lowry or Bradford methods. Crude protein in feed is nitrogen by Kjeldahl or Dumas multiplied by 6.25, which assumes 16 percent nitrogen.
Centrifugation separates by size and density. Relative centrifugal force (RCF, in multiples of g) = 1.118 x 10^-5 x radius in cm x rpm squared; at 3,000 rpm and 10 cm radius, RCF is about 1,006 g. Protocols should state RCF, not rpm, because rpm means different forces in different rotors.
Chromatography separates by partition between a mobile and a stationary phase. Ion-exchange chromatography separates by charge; gel filtration (size-exclusion) by size, with large molecules eluting first; affinity chromatography by specific binding, such as an antibody or a nickel column for histidine-tagged proteins. High-performance liquid chromatography (HPLC) is used for vitamins, drugs and amino acids; gas chromatography is the standard method for rumen VFA and fatty acid profiles.
Electrophoresis separates charged molecules in an electric field. Serum protein electrophoresis on agarose or by capillary methods gives albumin, alpha, beta and gamma globulin fractions; a narrow gamma spike suggests a monoclonal gammopathy such as multiple myeloma, and a broad increase suggests chronic antigenic stimulation. SDS-PAGE separates denatured proteins by size. Agarose gels separate DNA fragments. Isoelectric focusing separates by pI. Blotting transfers separated molecules to a membrane for probing: Southern for DNA, Northern for RNA and Western for protein.
Immunoassays use antibodies for specificity. ELISA links an antibody or antigen to an enzyme whose coloured product is read photometrically. They measure hormones such as cortisol, T4 and progesterone and detect infections. Species specificity matters: an assay validated for one species may not measure the hormone of another reliably. The polymerase chain reaction amplifies a target DNA sequence through repeated cycles of denaturation, primer annealing and extension by a heat-stable DNA polymerase, doubling the target each cycle in ideal conditions; reverse transcription PCR starts from RNA, and quantitative (real-time) PCR follows amplification with fluorescence.
Many laboratory errors happen before analysis. Haemolysis releases intracellular substances and interferes optically: it raises AST, LDH and, in species with high red-cell potassium such as horses, pigs and some dog breeds including the Akita, potassium. Lipaemia scatters light. EDTA contamination of serum chelates calcium and magnesium, lowering them and inhibiting ALP, and potassium EDTA raises potassium. Cells in unseparated blood keep using glucose, lowering it; fluoride oxalate tubes inhibit glycolysis (enolase) for glucose samples. Reference intervals are usually the central 95 percent of values from healthy animals, so 1 healthy animal in 20 falls outside for any one test; they depend on species, breed, age, physiological state, method and analyser, so each laboratory should establish or validate its own, following guidelines such as those of the American Society for Veterinary Clinical Pathology.
Learn each pathway as a set of decisions: which enzyme controls it, which vitamin it needs, which hormone switches it and where it happens. Then connect each to a disease you will meet later: thiamine and polioencephalomalacia, B12 and cobalt wasting, CPT-I and ketosis, VLDL export and fatty liver, taurine and feline cardiomyopathy. Practise the four standard calculations (Michaelis-Menten, Henderson-Hasselbalch, ATP yield, enzyme units) until you can do them without a formula sheet, because entrance and postgraduate papers reward speed.
Biochemistry is what you use every time you read a serum profile. Know which liver enzyme to trust in which species, why a dog's high ALP may be steroid induction, and how haemolysis, lipaemia, EDTA and delayed separation change results. In dairy work, use NEFA before calving and BHB after calving to monitor transition cows, choose cut-points from your own laboratory, and explain to farmers why body condition at calving and dry-matter intake drive ketosis and fatty liver. Treat ketosis and pregnancy toxaemia with glucogenic support at doses confirmed against the label.
At herd and system level the biochemistry becomes economics. Senior clinicians and consultants use herd BHB and NEFA prevalence, milk fat-to-protein ratio and milk urea to judge transition management, energy supply and rumen-degradable protein. They set mineral strategy by region, knowing where copper, cobalt, selenium and iodine are short or molybdenum and sulphur high. They advise laboratories and government programmes on test choice and interpretation, and they know that published cut-points and reference intervals vary, so decisions must rest on the laboratory and population in front of them.
Research frontiers include hepatic regulation of fatty acid oxidation versus esterification in transition cows, the roles of PPARs, AMPK and mTOR in mammary synthesis, insulin resistance and inflammation in early lactation, methane mitigation through redirected rumen hydrogen, and metabolomics of milk and blood as early warning of disease. A senior researcher designs experiments with isotopic tracers and proper kinetics, validates assays for each species, reports enzyme activity with method and temperature, and treats reference intervals and cut-points as population statistics that must be re-established rather than borrowed.
This manual does not give numerical reference intervals for any analyte, because they vary by species, age, method and laboratory; use the interval printed by the laboratory that ran the test. It does not give BHB or NEFA cut-points as fixed numbers for the same reason. Molecular biology and recombinant DNA technology are covered only as far as biochemistry needs them; detailed biotechnology belongs to the biotechnology subject. Porphyrias, detailed inborn errors of metabolism in animals, xenobiotic metabolism (cytochrome P450) and the biochemistry of exotic and aquatic species are only touched on. Drug doses for ketosis and pregnancy toxaemia are not given; confirm against the product label and your national formulary.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions. Every numerical answer was computed by script. Clinical reference intervals and ketosis cut-points vary by species, method and laboratory; use the interval of the laboratory that ran the test.
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