Learn — Biochemistry: enzymes, metabolism, acid–base & lab values

15 notes · 37 questions on the topic

Major anions and cations

Body water is about 60 per cent of body weight in an adult man, two-thirds of it inside the cells and one-third outside them as interstitial fluid and plasma, and each compartment is held electrically neutral by its own set of cations and anions. The distribution is not random and it is the whole basis of fluid therapy: sodium with chloride and bicarbonate dominates the extracellular fluid and therefore determines its volume and osmolality, while potassium with phosphate and protein dominates the intracellular fluid and therefore sets the resting membrane potential on which every heartbeat depends.

Extracellular fluid: sodium is the major cation at 135 to 145 mEq/L with chloride at 98 to 106 mEq/L and bicarbonate at 22 to 26 mEq/L as the major anions, plus small contributions from potassium, calcium, magnesium, phosphate, sulphate, organic acids and protein.
Intracellular fluid: potassium is the major cation at roughly 150 mEq/L, with magnesium the second, and organic phosphate and protein the major anions; the serum potassium of 3.5 to 5.0 mEq/L therefore represents only about 2 per cent of the body's potassium, which is why a normal level can coexist with a large total-body deficit.
Because the potassium gradient across the membrane sets the resting potential, small changes are dangerous: hyperkalaemia gives tall peaked T waves, a prolonged PR interval, loss of the P wave, a widening QRS and finally a sine wave and asystole, while hypokalaemia gives flattened T waves, ST depression, prominent U waves, arrhythmia and digoxin toxicity.
Sodium determines plasma osmolality and therefore where water goes; normal serum osmolality is about 285 to 295 mOsm/kg and can be calculated as twice the sodium plus the glucose divided by 18 plus the blood urea nitrogen divided by 2.8 when those are in mg/dL, and a measured value much higher than the calculated one signals an unmeasured osmole such as alcohol.
The anion gap is sodium minus the sum of chloride and bicarbonate, normally about 8 to 12 mEq/L, and it separates the two kinds of metabolic acidosis: a raised gap from added acid (the MUDPILES list — methanol, uraemia, diabetic ketoacidosis, propylene glycol, isoniazid or iron, lactic acidosis, ethylene glycol and salicylates) and a normal gap, hyperchloraemic acidosis from lost bicarbonate in diarrhoea, renal tubular acidosis or large volumes of saline.
Serum total calcium of about 8.5 to 10.5 mg/dL is roughly half bound to albumin, so a low albumin lowers the total without symptoms; it is the ionised fraction that matters, and it falls in alkalosis — which is why a hyperventilating patient develops tetany with a normal total calcium — and rises in acidosis.
Intravenous fluids are electrolyte prescriptions: 0.9 per cent sodium chloride carries 154 mEq/L each of sodium and chloride and in large volumes causes a hyperchloraemic acidosis; Ringer's lactate is closer to plasma and contains calcium, which is why it must never share a line with blood; and 5 per cent dextrose is effectively free water once the sugar is metabolised, so it does not stay in the vascular compartment.
Potassium is never given as an intravenous bolus or push — it is fatal; it is diluted and infused, conventionally at no more than 10 mEq per hour and at a concentration up to about 40 mEq/L through a peripheral line, with cardiac monitoring and central access for anything faster, and it is withheld until urine output is confirmed.
Magnesium is the silent partner: hypokalaemia and hypocalcaemia that will not correct despite repeated replacement are usually the result of an uncorrected hypomagnesaemia, so magnesium is checked and replaced first.

Urine specific gravity interpretation

Urine specific gravity is the ratio of the density of urine to the density of an equal volume of water, so it measures how much solute the kidney has packed into the water it excreted. It is the cheapest bedside index of hydration and of tubular concentrating ability, and it earns its place on the observation chart because a single value read alongside the urine output and the fluid balance separates the dehydrated patient who is conserving water from the patient whose kidney has lost the ability to conserve it at all.

The normal range on a random specimen is about 1.003 to 1.030, and a healthy first morning specimen after overnight fasting should be above about 1.020, since that is the test of whether the kidney can concentrate at all.
A value fixed at around 1.010 is isosthenuria — urine of the same concentration as plasma — and it means the tubule can neither concentrate nor dilute; it is the classic finding of established chronic kidney disease and of acute tubular necrosis.
A high specific gravity means water is being conserved or heavy solute is present: dehydration, vomiting, diarrhoea, fever, reduced renal perfusion in heart failure, the syndrome of inappropriate antidiuretic hormone, and glycosuria or proteinuria; radiographic contrast, dextran and mannitol raise it sharply and falsely because the molecules are large.
A low specific gravity means water is being lost: over-hydration and excessive intravenous fluid, diuretic therapy, chronic kidney disease, and above all diabetes insipidus, where a large output of very dilute urine below about 1.005 continues despite a rising serum sodium.
The discriminator between the two diabetes is written in the urine: polyuria with a high specific gravity and heavy glycosuria is diabetes mellitus, while polyuria with a persistently low specific gravity and no glucose is diabetes insipidus.
Specific gravity helps separate the causes of acute kidney injury — a prerenal, volume-responsive picture concentrates the urine above about 1.020 with a low urinary sodium, whereas intrinsic acute tubular necrosis gives a fixed specific gravity near 1.010 with a high urinary sodium.
Method matters: the reagent strip uses an ion-exchange colour reaction and reads only ionic solute, so it is relatively unaffected by glucose and contrast, while a refractometer or urinometer reads total solute and must be corrected — conventionally subtracting about 0.004 for each gram per decilitre of glucose and about 0.003 for each gram per decilitre of protein, and correcting for temperature.
Urine osmolality is the more precise measurement and is the one used for formal testing such as the water deprivation test, but specific gravity is what is available at the bedside and correlates closely enough when there is no heavy solute present.
The reading is only interpretable in context, so it is charted with the hourly or daily urine output, the fluid balance, the daily weight (one kilogram of weight change is about one litre of fluid) and the serum sodium — never read alone.

Ketone bodies

Ketone bodies are the three small water-soluble fuels — acetoacetate, beta-hydroxybutyrate and acetone — made in liver mitochondria from acetyl-CoA by way of HMG-CoA when fat oxidation runs faster than the citric acid cycle can consume its acetyl-CoA, as it does in starvation, prolonged exercise and insulin deficiency. They matter because they are a physiological adaptation that lets the brain run on fat-derived fuel and spares muscle protein, but the same pathway unrestrained by insulin produces the organic acid load of diabetic ketoacidosis, so the nurse must be able to tell useful ketosis from lethal ketoacidosis.

Ketogenesis happens only in the liver, but the liver cannot use ketone bodies because it lacks the enzyme thiophorase that reactivates acetoacetate; the consumers are the brain, skeletal muscle, heart and renal cortex, and after two to three days of fasting the brain derives a large share of its energy from ketones instead of glucose.
Beta-hydroxybutyrate is quantitatively the major ketone body in diabetic ketoacidosis, acetoacetate is the parent compound, and acetone is a non-acidic breakdown product that is volatile and blown off by the lungs — which is the fruity or nail-polish smell on the breath.
The urine dipstick uses a nitroprusside reaction that detects acetoacetate and acetone but not beta-hydroxybutyrate, so it underestimates ketosis at the start and may appear to rise during treatment as beta-hydroxybutyrate is converted back to acetoacetate; a point-of-care blood beta-hydroxybutyrate meter is the correct measurement and the one used to judge resolution.
Diabetic ketoacidosis is diagnosed by three findings together, not by ketones alone: hyperglycaemia (usually above 250 mg/dL, though it can be near-normal in euglycaemic ketoacidosis with SGLT2 inhibitors or in pregnancy), ketonaemia with blood beta-hydroxybutyrate at or above about 3 mmol/L or significant ketonuria, and metabolic acidosis with a pH below about 7.3 or a bicarbonate below about 15 to 18 mEq/L, with a raised anion gap.
The cause is absolute insulin deficiency with an excess of the counter-regulatory hormones glucagon, cortisol, catecholamines and growth hormone, which together drive lipolysis and hepatic ketogenesis; the trigger is almost always identifiable — infection, missed insulin doses, a first presentation of type 1 diabetes, myocardial infarction, trauma or surgery — and finding it is part of the treatment.
Ketosis without acidosis is a different animal: starvation and post-exercise ketosis, the ketogenic diet used in refractory childhood epilepsy, and ketonuria in hyperemesis gravidarum all produce ketones with a normal pH, while alcoholic ketoacidosis produces acidosis with a normal or low glucose.
Treatment order in ketoacidosis is fluid first, then insulin, then potassium and glucose: isotonic saline restores circulating volume, a fixed-rate intravenous insulin infusion switches off ketogenesis, and dextrose is added once the glucose falls to about 200 to 250 mg/dL so the insulin can be continued until the ketones clear — because it is the ketones, not the glucose, that are being treated.
Potassium is the number that kills: total body potassium is severely depleted even when the serum level reads normal or high, and insulin drives it into the cells, so potassium is added to the fluids once the level is known and urine is flowing, and insulin is withheld until the potassium is above about 3.3 mEq/L if it is low to start with.
Monitoring is hourly and relentless — capillary glucose every hour, blood ketones and venous bicarbonate or pH, potassium every two hours at the beginning, strict fluid balance, continuous cardiac monitoring and neurological observations, since cerebral oedema is the leading cause of death in children with ketoacidosis and is heralded by headache, irritability, a falling conscious level, bradycardia and rising blood pressure.

Omega-3 PUFA

Omega-3 fatty acids are polyunsaturated fatty acids whose first double bond lies three carbons from the methyl or omega end of the chain, the parent being alpha-linolenic acid, an essential fatty acid the human body cannot synthesise, from which eicosapentaenoic acid and docosahexaenoic acid are made inefficiently. They matter because they compete with the omega-6 arachidonic acid as the precursor of eicosanoids, so a diet richer in omega-3 shifts prostaglandin and leukotriene production towards less inflammatory and less thrombotic species, and because docosahexaenoic acid is a structural lipid of the retina and the developing brain.

The two essential fatty acids are linoleic acid, an omega-6, and alpha-linolenic acid, an omega-3; deficiency causes a dry scaly dermatitis, poor wound healing and impaired growth, and it is seen in long-term fat-free parenteral nutrition and severe fat malabsorption.
Indian sources are fatty fish such as mackerel, sardine, hilsa and rohu, along with flaxseed or linseed, mustard oil, soybean oil, walnuts and fenugreek seeds, while eicosapentaenoic acid and docosahexaenoic acid in their ready-made form come essentially from marine fish and from breast milk.
Docosahexaenoic acid is concentrated in the photoreceptors of the retina and in the grey matter, and most of it is laid down in the last trimester and the first two years of life, which is one of the arguments for maternal nutrition in late pregnancy and for exclusive breastfeeding.
The recognised therapeutic effect is a substantial fall in serum triglycerides at pharmacological doses, with smaller effects on blood pressure, platelet aggregation and inflammation; the evidence is strong for eating fish about twice a week and much weaker for taking capsules, so supplements are prescribed for hypertriglyceridaemia rather than handed out for general prevention.
The balance matters as much as the amount: modern diets built on refined sunflower or safflower oil carry a very high ratio of omega-6 to omega-3, and dietary advice aims to bring it down towards the order of 5 to 10 to 1 by adding mustard oil, flaxseed, nuts and fish rather than by removing all omega-6.
Polyunsaturated oils oxidise and turn rancid on repeated heating, which destroys the fatty acid and generates harmful products, so they are not suitable for repeated deep frying and should be stored away from light and heat.
Cautions to teach: high doses prolong bleeding time and matter in a patient on warfarin, a direct oral anticoagulant or an antiplatelet drug and before surgery; capsules commonly cause fishy eructation, nausea and loose stools, which are reduced by taking them with food or freezing the capsule; and large predatory fish carry methylmercury, so pregnant and breastfeeding women choose small fish and limit intake.

Triglyceride transport (VLDL)

Lipids are insoluble in plasma, so they are carried inside lipoproteins — spherical particles with a core of triglyceride and cholesteryl ester wrapped in a shell of phospholipid, free cholesterol and apoproteins that act both as structural scaffolding and as addresses that switch enzymes and receptors on. Very low density lipoprotein is the particle by which the liver exports triglyceride it has made itself, and the sequence chylomicron, VLDL, IDL, LDL and HDL is worth knowing precisely because every lipid-lowering drug and every lipid profile the nurse sends is an intervention somewhere along it.

The five classes run from largest and least dense to smallest and most dense as chylomicron, very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein and high density lipoprotein; density rises as the protein content rises and the lipid content falls.
Chylomicrons carry exogenous, dietary triglyceride from the intestine through the lymphatics and thoracic duct into the blood and carry apoprotein B-48, whereas very low density lipoprotein carries endogenous triglyceride made in the liver and carries apoprotein B-100 — the single discriminator most often asked.
Lipoprotein lipase sits on the capillary endothelium of adipose tissue, skeletal muscle and heart, is activated by apoprotein C-II and released into the plasma by heparin, and hydrolyses the triglyceride of both chylomicrons and very low density lipoprotein so the fatty acids can be taken up; a hereditary deficiency of this enzyme or of apoprotein C-II causes massive chylomicronaemia with recurrent pancreatitis.
As triglyceride is stripped away, very low density lipoprotein becomes intermediate density lipoprotein and then low density lipoprotein, which carries most of the plasma cholesterol to the tissues and is taken up by the low density lipoprotein receptor; a defect in that receptor is familial hypercholesterolaemia, an autosomal dominant condition with tendon xanthomas, corneal arcus in youth and premature coronary disease.
Low density lipoprotein is called the bad cholesterol because when it is oxidised it is engulfed by macrophage scavenger receptors to form the foam cells of an atheromatous plaque, while high density lipoprotein is the good one because, carrying apoprotein A-I and activating lecithin cholesterol acyltransferase, it performs reverse cholesterol transport back to the liver.
The desirable values quoted in Indian practice are a total cholesterol below 200 mg/dL, a low density lipoprotein cholesterol below 100 mg/dL and lower still in established coronary disease, a triglyceride below 150 mg/dL, and a high density lipoprotein above 40 mg/dL in men and 50 mg/dL in women.
Low density lipoprotein cholesterol is usually calculated rather than measured, by the Friedewald formula — total cholesterol minus high density lipoprotein cholesterol minus triglyceride divided by five — which is valid only on a fasting sample and only when the triglyceride is below about 400 mg/dL, so a very high triglyceride makes the reported figure meaningless.
The drugs map onto the pathway: statins inhibit HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis, and work chiefly by up-regulating hepatic low density lipoprotein receptors; fibrates and high-dose omega-3 lower triglyceride; ezetimibe blocks intestinal absorption; and bile acid sequestrants interrupt the enterohepatic circulation.

Prokaryotic vs eukaryotic cell

A prokaryotic cell has no membrane-bound nucleus and no membrane-bound organelles: its single circular chromosome lies naked in the cytoplasm in a region called the nucleoid, and bacteria and archaea are the only prokaryotes. A eukaryotic cell keeps its multiple linear chromosomes inside a true nucleus bounded by a nuclear envelope and compartmentalises its work into mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes; the distinction is not academic, because every structural difference between the two is a point at which an antimicrobial drug can poison the microbe and spare the patient.

Ribosomes are the classic difference: the prokaryotic ribosome is 70S, built from 30S and 50S subunits, while the eukaryotic cytoplasmic ribosome is 80S from 40S and 60S subunits — which is why aminoglycosides and tetracyclines (30S) and macrolides, clindamycin, chloramphenicol and linezolid (50S) can kill bacteria without stopping human protein synthesis.
The exception that explains the toxicity is that human mitochondria contain their own 70S ribosomes, so ribosome-targeting antibiotics do reach a human target: chloramphenicol causes bone marrow suppression and the grey baby syndrome, and prolonged linezolid causes cytopenias, lactic acidosis and optic neuropathy.
Bacteria have a peptidoglycan cell wall that human cells lack entirely, which is the basis of the selective toxicity of penicillins, cephalosporins and vancomycin; mycoplasma has no wall and is therefore intrinsically resistant to them, while fungi have a chitin and glucan wall and an ergosterol membrane, targeted by echinocandins and azoles respectively.
Genetic material differs in form as well as location: a prokaryote has one circular double-stranded chromosome without histones plus plasmids that carry antibiotic resistance genes and pass between bacteria by conjugation, whereas a eukaryote has many linear chromosomes wound on histones, with introns that must be spliced out and messenger RNA that is capped and polyadenylated before it leaves the nucleus.
Because a prokaryote has no nuclear envelope, transcription and translation happen simultaneously on the same messenger RNA, and its messenger RNA is polycistronic, carrying several genes in one operon such as the lac operon; in a eukaryote the two processes are separated in space and time and each messenger RNA carries a single gene.
Prokaryotes divide by simple binary fission with no spindle and no mitosis, which is why they can double in about 20 minutes, while eukaryotes divide by mitosis or meiosis using a microtubule spindle — the reason vinca alkaloids and taxanes are anticancer drugs and not antibiotics.
Other exploitable differences are bacterial DNA gyrase and topoisomerase IV, which the fluoroquinolones inhibit, and bacterial de novo folate synthesis, which sulphonamides and trimethoprim block — humans take folate ready-made from the diet and so are unaffected.
Size and complexity differ by an order of magnitude, a bacterium being roughly 1 to 10 micrometres against a typical animal cell of 10 to 100 micrometres, and viruses belong to neither group: they have no ribosomes and no metabolism of their own and must use the host cell's machinery, which is why antibacterial antibiotics are useless against them.

Sequence of mitosis

Mitosis is the division of a somatic cell nucleus that produces two genetically identical diploid daughter cells, each keeping the full complement of 46 chromosomes, and it occupies the M phase of a cell cycle whose remaining stages — G1, S and G2, together called interphase — are spent growing and copying the DNA. It matters clinically because the tissues that cycle fastest are the bone marrow, the gastrointestinal mucosa, the hair follicle and the gonad, and those are exactly the tissues that cytotoxic drugs and radiation damage, so the whole pattern of chemotherapy toxicity is written into this sequence.

Interphase comes first and occupies most of the cycle: in G1 the cell grows and decides whether to divide, in S phase the DNA is replicated so that each chromosome now consists of two sister chromatids joined at the centromere, and in G2 the cell checks the copy and builds what the spindle will need; cells that leave the cycle enter the resting G0 state, where neurons and cardiac muscle stay permanently.
Prophase: the chromatin condenses into visible chromosomes each of two chromatids, the nucleolus disappears, the centrosomes move to opposite poles and the microtubule spindle forms; at the end of prophase the nuclear envelope breaks down and the spindle fibres attach to the kinetochore of each chromosome.
Metaphase: the chromosomes are pulled into a single line on the equatorial plate and are at their most condensed, which is why this is the stage used for karyotyping and why colchicine, which arrests cells in metaphase, is added to the culture before a chromosome study.
Anaphase: the centromeres split and the sister chromatids are dragged to opposite poles as separate chromosomes; failure of a pair to separate here is non-disjunction, which leaves one daughter cell with an extra chromosome and the other with one too few, and is the mechanism behind trisomy and monosomy.
Telophase: the chromatids reach the poles and decondense, the nuclear envelope and nucleolus re-form around each set and the spindle disappears; cytokinesis then pinches the cytoplasm in two with a contractile ring of actin and myosin, giving two diploid cells. The order is remembered as prophase, metaphase, anaphase, telophase.
Mitosis is not meiosis: mitosis is one division producing two identical diploid somatic cells, whereas meiosis is two successive divisions producing four genetically different haploid gametes, with crossing over between homologous chromosomes in prophase I, and it is non-disjunction in maternal meiosis that underlies the rising risk of Down syndrome with maternal age.
The cycle is policed at checkpoints at G1 to S, at G2 to M and at the spindle; p53, the guardian of the genome, halts a damaged cell at G1 for repair or sends it to apoptosis, and loss of p53 is the commonest mutation in human cancer, which is fundamentally a disease of unregulated cell division.
Cytotoxic drugs are classified by where they strike: vinca alkaloids such as vincristine and vinblastine bind tubulin and prevent the spindle forming, arresting the cell in metaphase, taxanes such as paclitaxel do the opposite by stabilising microtubules so they cannot disassemble, methotrexate and cytarabine act in S phase, and alkylating agents work at any point in the cycle.
Because the drugs hit dividing cells indiscriminately, the predictable toxicities are myelosuppression with a neutrophil nadir usually about 7 to 14 days after a dose, mucositis and diarrhoea from the gut lining, alopecia from the hair follicle and infertility from the gonad — and the nursing plan for a patient on chemotherapy is built around that timetable.

Mitochondria — powerhouse

The mitochondrion is the double-membraned organelle in which the citric acid cycle, the beta-oxidation of fatty acids and oxidative phosphorylation take place, and it supplies almost all the ATP that a cell uses. It is called the powerhouse for that reason, but its clinical importance is wider: it holds its own maternally inherited circular DNA, it releases the cytochrome c that triggers apoptosis, and it is the exact target of cyanide, carbon monoxide and the hypoxia of shock — so when a cell dies of poisoning or of low oxygen, this is where it dies.

The outer membrane is smooth and freely permeable to small molecules through porin channels, while the inner membrane is thrown into cristae, is impermeable and carries the four complexes of the electron transport chain and ATP synthase; the enclosed matrix holds the citric acid cycle and beta-oxidation enzymes, mitochondrial DNA and mitochondrial ribosomes.
Because the inner membrane is impermeable, everything needs a carrier: long-chain fatty acids enter on the carnitine shuttle, and the reducing equivalents of cytosolic NADH enter through the malate-aspartate shuttle in liver, kidney and heart or the glycerol phosphate shuttle in muscle and brain.
The number of mitochondria matches the energy demand of the tissue, so cardiac muscle, hepatocytes, renal tubular cells and skeletal muscle are packed with them, while the mature red blood cell has none at all — which is why it depends entirely on anaerobic glycolysis and can use neither fatty acids nor ketone bodies.
Mitochondrial DNA is a small circular double-stranded molecule that codes for a handful of respiratory-chain polypeptides and its own transfer and ribosomal RNAs; it is inherited exclusively from the mother because the sperm's mitochondria are destroyed after fertilisation, so an affected mother passes the disease to all her children while an affected father passes it to none — the pedigree pattern that identifies conditions such as Leber's hereditary optic neuropathy and MELAS.
Oxidative phosphorylation works by chemiosmosis: complexes I to IV pass electrons along and pump protons out of the matrix, creating an electrochemical gradient across the inner membrane, and complex V, the ATP synthase, lets those protons back in and uses the energy to make ATP; complete oxidation of one molecule of glucose yields about 30 to 32 ATP by current estimates.
The inhibitors are examined as a set: rotenone and amobarbital block complex I, antimycin A complex III, cyanide, carbon monoxide, hydrogen sulphide and azide block complex IV or cytochrome c oxidase, and oligomycin blocks ATP synthase; the uncouplers 2,4-dinitrophenol and thyroxine in excess, and the physiological uncoupling protein thermogenin in the brown adipose tissue of the newborn, dissipate the gradient as heat instead of ATP.
Cyanide poisoning is the clinical face of this pathway: the tissues cannot use the oxygen delivered to them, so the patient has a normal or high pulse oximeter reading, unusually red venous blood with a high venous oxygen saturation, a severe lactic acidosis and rapid loss of consciousness, and the classic setting is smoke inhalation from an enclosed fire.
Mitochondria also start the intrinsic pathway of apoptosis by releasing cytochrome c, buffer intracellular calcium, carry out the first step of the urea cycle and of haem synthesis, and are the site of the toxicity of several drugs — metformin-associated lactic acidosis, propofol infusion syndrome and the mitochondrial toxicity of nucleoside analogue antiretrovirals.

Cytoskeleton

The cytoskeleton is the three-dimensional network of protein filaments that gives a cell its shape and mechanical strength, anchors its organelles, moves material through its cytoplasm and drives its division and locomotion. It is made of three filament families of different diameter and different protein — microfilaments of actin, intermediate filaments of tissue-specific proteins, and microtubules of tubulin — and the distinction is clinically useful because intermediate filaments identify the tissue of origin of a tumour and microtubules are the target of a whole family of drugs.

Microfilaments are the thinnest, about 7 nanometres, and are polymers of actin; with myosin they produce muscle contraction, they form the core of the microvilli of the intestinal brush border, and they drive cell crawling, phagocytosis and the contractile ring that pinches the cell in two during cytokinesis.
Intermediate filaments are about 10 nanometres and are purely structural, and because each tissue makes its own type they are used in immunohistochemistry to say where an undifferentiated tumour came from: cytokeratin in epithelium and therefore in carcinoma, vimentin in mesenchymal tissue and sarcoma, desmin in muscle, glial fibrillary acidic protein in glial cells, neurofilaments in neurons and lamins lining the inner surface of the nuclear envelope.
Keratin, the intermediate filament of skin, hair and nail, is the example to remember, and mutations in the keratin genes of the basal keratinocytes cause epidermolysis bullosa simplex, in which the skin blisters with minor friction.
Microtubules are the largest at about 25 nanometres, are hollow tubes built from dimers of alpha and beta tubulin, and they form the mitotic spindle, the centrioles, the transport tracks along which kinesin carries cargo away from the cell body and dynein carries it back, and the axoneme of cilia and flagella.
The axoneme of a motile cilium or a sperm flagellum has the nine-plus-two arrangement — nine outer doublets around a central pair — with dynein arms that produce the sliding; a centriole, by contrast, is nine triplets with no central pair.
A defect in the dynein arms causes primary ciliary dyskinesia, and its classic form is Kartagener's syndrome — situs inversus with dextrocardia, chronic sinusitis and bronchiectasis, with male infertility from immotile sperm — because the same motor moves the respiratory cilia, the sperm tail and the embryonic node that decides left-right asymmetry.
Microtubules are the target of an unusually large number of drugs: colchicine, used in gout, prevents polymerisation and so blocks neutrophil migration into the joint; vinca alkaloids prevent the spindle from assembling; taxanes stabilise microtubules so they cannot disassemble; griseofulvin acts on fungal microtubules; and albendazole and mebendazole bind parasite tubulin.
The bedside consequence of blocking microtubules is that everything which depends on them stops together, which is why vincristine causes a dose-limiting peripheral neuropathy with loss of the ankle jerk, numb fingers, foot drop and, because it also paralyses the microtubule-dependent gut, severe constipation and paralytic ileus.

TCA cycle

The tricarboxylic acid cycle, also called the citric acid or Krebs cycle, is the final common oxidative pathway of carbohydrate, fat and protein, and it runs in the mitochondrial matrix, where the two-carbon acetyl group of acetyl-CoA is oxidised completely to two molecules of carbon dioxide and its energy is captured as reduced coenzymes. It matters because it is amphibolic — the same intermediates that are burnt for energy are also the raw material for gluconeogenesis, haem, amino acids and fatty acids — and because although the cycle itself uses no oxygen, it stops the moment oxygen runs out, which is why a shocked patient's lactate rises.

The whole cycle takes place in the mitochondrial matrix with one exception that is regularly asked: succinate dehydrogenase is bound to the inner mitochondrial membrane and is also complex II of the electron transport chain.
The entry step is irreversible and vitamin-dependent: pyruvate dehydrogenase converts pyruvate to acetyl-CoA and requires five cofactors — thiamine pyrophosphate, lipoic acid, coenzyme A from pantothenic acid, FAD from riboflavin and NAD from niacin — which is precisely why thiamine deficiency produces lactic acidosis and neurological disease.
Acetyl-CoA condenses with oxaloacetate to form citrate, and one complete turn of the eight-step cycle releases two molecules of carbon dioxide and yields three NADH, one FADH2 and one GTP, the GTP being the only substrate-level phosphorylation in the pathway.
Feeding those coenzymes into the respiratory chain gives roughly 10 ATP per turn on current estimates of about 2.5 ATP per NADH and 1.5 per FADH2; older textbooks that count 3 and 2 give 12 ATP per turn, so quote the convention of the book you are answering from.
Regulation is by the energy state of the cell: isocitrate dehydrogenase is the main rate-limiting enzyme, activated by ADP and inhibited by ATP and NADH, with citrate synthase and alpha-ketoglutarate dehydrogenase also controlled, so the cycle slows when the cell is already rich in ATP.
The cycle is amphibolic, not merely catabolic: citrate is exported to the cytosol to supply acetyl units for fatty acid synthesis, alpha-ketoglutarate and oxaloacetate are transaminated to glutamate and aspartate, succinyl-CoA begins haem synthesis, and oxaloacetate and malate feed gluconeogenesis — which is why intermediates must be replaced by anaplerotic reactions, chiefly pyruvate carboxylase, a biotin-dependent enzyme.
No oxygen is consumed within the cycle itself, but NAD+ and FAD are regenerated only by the electron transport chain, so in hypoxia or shock the cycle halts, pyruvate is diverted to lactate and a lactic acidosis develops — the biochemical explanation of the lactate that guides resuscitation.
The classic inhibitors are fluoroacetate, which becomes fluorocitrate and blocks aconitase, arsenite, which poisons the lipoic-acid-dependent pyruvate and alpha-ketoglutarate dehydrogenases, and malonate, the textbook competitive inhibitor of succinate dehydrogenase.

Lactose

Lactose is the sugar of milk, a disaccharide of glucose linked to galactose by a beta-1,4 glycosidic bond, and it is the only significant carbohydrate in human and animal milk. It must be split by lactase at the brush border of the small-intestinal villus before either sugar can be absorbed, and because lactase sits at the very tip of the villus and is the first enzyme lost when the mucosa is damaged, unsplit lactose in the lumen — drawing in water and fermented by colonic bacteria — is one of the commonest reasons a child's diarrhoea drags on after the infection has gone.

Lactose is glucose plus galactose in a beta-1,4 link, in contrast to sucrose, which is glucose plus fructose, and maltose, which is glucose plus glucose; lactose and maltose are reducing sugars and give a positive Benedict's test, while sucrose is non-reducing.
Primary adult-type hypolactasia is the normal, genetically programmed fall in lactase activity after weaning and affects the majority of adults in Asia and Africa; it is lactase persistence into adulthood, seen in northern European and some pastoral populations, that is the unusual state.
Secondary lactose intolerance is the form seen on the wards: any insult that flattens the villous tip — rotavirus and other gastroenteritis, giardiasis, coeliac disease, severe malnutrition, small bowel surgery — removes lactase first and restores it last, so it is temporary and recovers over weeks.
The symptoms come from osmosis and fermentation, not from immunity: bloating, borborygmi, cramps, flatus and a watery, frothy, acidic stool that excoriates the perianal skin, beginning about 30 minutes to 2 hours after milk and settling when milk is withdrawn, with no rash, wheeze or vomiting.
Cow's milk protein allergy is the differential that must not be confused with it — that is an immune reaction to the protein, not the sugar, and can cause vomiting, blood or mucus in the stool, eczema, urticaria, wheeze and even anaphylaxis, and it requires complete elimination of cow's milk protein rather than a reduction in lactose.
Tests are simple: a stool pH below about 5.5 with reducing substances present in a child, a lactose hydrogen breath test, a lactose tolerance test in which the blood glucose fails to rise adequately after an oral load, and, rarely, small-bowel biopsy with disaccharidase assay.
Management is reduction rather than elimination: breastfeeding is continued in an infant with diarrhoea and is never stopped for suspected lactose intolerance, most adults tolerate small amounts of milk taken with a meal, curd, yoghurt and hard cheese are well tolerated because their bacteria have already broken down much of the lactose, and lactase drops or lactose-free formula are used when they are genuinely needed — always with attention to replacing the calcium and vitamin D.
Galactosaemia is an entirely different and far more serious disease that must not be confused with lactose intolerance: it is a deficiency of galactose-1-phosphate uridyl transferase presenting in the first weeks with vomiting, jaundice, hepatomegaly, cataract, Escherichia coli sepsis and failure to thrive, and in it all milk including breast milk must be stopped and a lactose-free, galactose-free formula given lifelong.

DNA structure

DNA is the double-stranded helical polymer that stores the genetic information of the cell, built from nucleotides each made of a deoxyribose sugar, a phosphate and one of four bases — the purines adenine and guanine and the pyrimidines cytosine and thymine. In the Watson and Crick model the two strands run antiparallel around a common axis and are held together by hydrogen bonds between complementary bases, and it is that complementarity — the fact that each strand carries the full information needed to rebuild the other — which makes replication, transcription and every modern genetic test possible.

The backbone is an alternating chain of deoxyribose and phosphate joined by 3-prime to 5-prime phosphodiester bonds, with the bases pointing inwards; the two strands are antiparallel, one running 5-prime to 3-prime and the other 3-prime to 5-prime.
Base pairing is fixed: adenine pairs with thymine through two hydrogen bonds and guanine with cytosine through three, so a purine always faces a pyrimidine and the helix keeps a constant width, and a sequence rich in guanine and cytosine needs a higher temperature to separate.
Chargaff's rule follows from that pairing — in any double-stranded DNA the amount of adenine equals thymine and guanine equals cytosine, so total purines equal total pyrimidines — and it is the basis of the calculation questions in which one base percentage is given and the rest must be deduced.
The common B form is a right-handed helix with about 10 base pairs per complete turn, a rise of about 0.34 nanometres per base pair and a diameter of about 2 nanometres, with major and minor grooves into which regulatory proteins fit; A-DNA and the left-handed Z-DNA are the alternative conformations.
Human DNA is found in two places: linear chromosomes in the nucleus, packed by winding about 147 base pairs around a histone octamer to form each nucleosome, and a small circular molecule in the mitochondrion; RNA differs in having ribose instead of deoxyribose, uracil in place of thymine and a single strand.
Replication is semiconservative, so each daughter molecule keeps one parental strand — the Meselson and Stahl result — and because DNA polymerase can only add nucleotides in the 5-prime to 3-prime direction, one strand is copied continuously as the leading strand and the other in short Okazaki fragments that are joined by DNA ligase.
Heating separates the strands and cooling lets them find their partners again, and this reversible denaturation is what the polymerase chain reaction and every hybridisation-based test exploit to amplify or detect a specific sequence.
The repair systems that protect the molecule are themselves examined through the diseases that follow their failure — xeroderma pigmentosum from defective nucleotide excision repair, with extreme sun sensitivity and early skin cancer, and Lynch syndrome from defective mismatch repair — while bacterial DNA gyrase is the target of the fluoroquinolones and DNA itself is the target of alkylating agents and ionising radiation.

Chromosome number (46)

Every normal human somatic cell carries 46 chromosomes arranged as 23 pairs — 22 pairs of autosomes and one pair of sex chromosomes, XX in the female and XY in the male — with one member of each pair inherited from each parent. Gametes carry the haploid number of 23 so that fertilisation restores 46, and the number matters clinically because the commonest congenital disorders a nurse will meet, from Down syndrome to Turner syndrome, are errors in this count produced by a single failure of chromosomes to separate during cell division.

The somatic diploid number is 46 and the gametic haploid number is 23; a normal karyotype is written 46,XX for a female and 46,XY for a male, and it is prepared from cultured lymphocytes arrested in metaphase, where the chromosomes are most condensed, then arranged by size and centromere position.
Non-disjunction — the failure of a chromosome pair or of sister chromatids to separate — leaves one cell with an extra chromosome and the other short of one, producing the trisomies and monosomies; the risk of maternal meiotic non-disjunction rises steeply with maternal age.
Down syndrome is trisomy 21, written 47,XX,+21 or 47,XY,+21, and presents with hypotonia, upslanting palpebral fissures with epicanthic folds, a flat nasal bridge, a protruding tongue, a single transverse palmar crease and a wide sandal gap, together with congenital heart disease, duodenal atresia, hypothyroidism, hearing loss and atlantoaxial instability; about 95 per cent is free trisomy from non-disjunction, a small proportion is a Robertsonian translocation — the form that can recur and therefore requires parental karyotyping — and a few per cent are mosaic.
Edwards syndrome is trisomy 18, with clenched hands and overlapping fingers, rocker-bottom feet, micrognathia and a prominent occiput; Patau syndrome is trisomy 13, with cleft lip and palate, microphthalmia, polydactyly and midline brain defects; both carry a very short life expectancy, and telling the three trisomies apart by their hands and faces is a standard examination question.
Sex chromosome aneuploidies: Klinefelter syndrome is 47,XXY, presenting after puberty with tall stature, small firm testes, gynaecomastia and infertility, while Turner syndrome is 45,X — the only monosomy compatible with life — with short stature, a webbed neck, widely spaced nipples, lymphoedema of the hands and feet in the newborn, coarctation of the aorta or a bicuspid aortic valve, and streak ovaries with primary amenorrhoea.
Structural abnormalities are counted separately from numerical ones and include deletion (the 5p deletion of cri-du-chat syndrome with its cat-like cry), translocation (the t(9;22) Philadelphia chromosome of chronic myeloid leukaemia), inversion, duplication and ring chromosomes, along with the trinucleotide repeat expansion of fragile X syndrome, the commonest inherited cause of intellectual disability.
The Barr body is the inactivated X chromosome seen as a dark mass at the nuclear margin, and the number of Barr bodies is always one fewer than the number of X chromosomes — none in a normal male, one in a normal female and one in a 47,XXY male.
Antenatal detection uses screening tests — first-trimester nuchal translucency with maternal serum markers, and cell-free fetal DNA — followed by the diagnostic tests, chorionic villus sampling at about 11 to 13 weeks and amniocentesis at about 15 to 18 weeks, each carrying a small procedure-related risk of miscarriage; in India the Pre-Conception and Pre-Natal Diagnostic Techniques Act permits these tests for genetic indications but makes disclosure of the fetal sex a criminal offence, and that prohibition binds the nurse as much as the doctor.

Mutation types

A mutation is a permanent, heritable change in the nucleotide sequence of DNA, ranging from the substitution of a single base to the loss or rearrangement of a whole segment of a chromosome. The class of mutation matters more than the fact of it, because whether a protein is unchanged, altered by one amino acid, cut short or destroyed entirely is what decides whether the patient is a healthy carrier or has a lethal disease — and because the same classes, arising in somatic cells, are how cancer begins.

A point mutation replaces one base with another and is called a transition when a purine replaces a purine or a pyrimidine replaces a pyrimidine, and a transversion when a purine and a pyrimidine are exchanged.
A point mutation in a coding region has three possible outcomes: silent, where the new codon still specifies the same amino acid because the code is degenerate, most often at the third base; missense, where a different amino acid is inserted — sickle cell disease, in which valine replaces glutamate at the sixth residue of the beta globin chain, is the standard example; and nonsense, where the codon becomes a stop signal and the protein is truncated and usually useless.
An insertion or deletion of bases in a number not divisible by three shifts the reading frame, so every codon after that point is misread and a premature stop usually follows; by contrast an in-frame deletion of exactly three bases removes a single amino acid and leaves the rest of the protein intact, which is what the delta-F508 deletion of cystic fibrosis does.
Mutations at a splice site prevent an intron being removed correctly and are a common molecular cause of beta thalassaemia, showing that a change outside the coding sequence can be as damaging as one within it.
Trinucleotide repeat expansions are a class of their own — CGG in fragile X syndrome, CAG in Huntington's disease, CTG in myotonic dystrophy and GAA in Friedreich's ataxia — and they show anticipation, in which the repeat lengthens between generations so the disease begins earlier and more severely in each successive one.
Larger changes are described at chromosome level as deletion, duplication, inversion, translocation and ring formation, and these are what a karyotype or a microarray detects, whereas a point mutation needs sequencing.
A germline mutation is present in the gametes and is passed to the next generation, while a somatic mutation affects only the cells descended from the one in which it arose — it cannot be inherited, but the stepwise accumulation of somatic mutations in oncogenes and tumour suppressor genes is exactly how a cancer develops.
Mutations arise spontaneously from errors in replication and from the chemical decay of bases, or are induced by mutagens — ionising radiation, ultraviolet light which forms thymine dimers, and chemicals such as alkylating agents, nitrosamines, benzene and aflatoxin — and it is these induced mutations that radiation protection and safe cytotoxic handling exist to prevent.

Essential amino acids

Essential or indispensable amino acids are the nine that the human body cannot synthesise at all, or cannot make fast enough to meet its needs, so they must be supplied ready-made in the diet: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, leucine and lysine. They matter because protein quality, not merely the number of grams eaten, is what determines growth, wound healing and immune competence — and because the Indian cereal-and-pulse plate is a practical solution to exactly this problem.

The nine essential amino acids are remembered as PVT TIM HaLL — phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, leucine and lysine — with histidine particularly indispensable in infancy and arginine regarded as semi-essential during growth.
Some amino acids are conditionally essential, meaning the body can normally make them but cannot make enough under stress: cysteine is made from methionine and tyrosine from phenylalanine, and cysteine, tyrosine, arginine, glutamine, proline and glycine all become essential in prematurity, sepsis, major trauma, burns and liver disease — while in phenylketonuria tyrosine is permanently essential because the enzyme that makes it is missing.
Cereals and pulses have opposite weaknesses, and this is the single most useful fact in Indian diet counselling: cereals such as rice, wheat and maize are limited by their low lysine but are relatively rich in methionine, whereas pulses such as dal, rajma and chana are limited by their low methionine but rich in lysine, so a mixture of roughly four parts cereal to one part pulse gives a protein of far higher quality than either alone.
Protein quality is measured as biological value, net protein utilisation and the digestibility-corrected amino acid score; egg protein has the highest biological value and is taken as the reference against which others are compared, milk and meat are close behind, and cereal protein is well below them, with soy the best of the plant proteins.
The daily protein requirement of a healthy adult is of the order of 0.8 to 1 gram per kilogram of body weight, and it rises in pregnancy, lactation, childhood growth, burns, sepsis, trauma, pressure injury and dialysis, while it is restricted only on specific medical instruction in advanced kidney disease.
Essential amino acids are also the precursors of signalling molecules that are asked about directly: tryptophan gives serotonin, melatonin and niacin; phenylalanine gives tyrosine, which gives dopamine, noradrenaline, adrenaline, thyroxine and melanin; histidine gives histamine; methionine gives S-adenosylmethionine, the universal methyl donor; and glycine with succinyl-CoA begins haem synthesis.
Leucine, isoleucine and valine are the branched-chain amino acids and are metabolised mainly in muscle rather than in liver; leucine and lysine are the only two purely ketogenic amino acids, meaning they cannot be converted into glucose.
The inborn errors of amino acid metabolism are examined as a set: phenylketonuria from phenylalanine hydroxylase deficiency, with a mousy odour, fair hair and skin and severe intellectual disability if untreated, detected by newborn screening and treated with a lifelong low-phenylalanine diet; maple syrup urine disease from a block in branched-chain keto acid oxidation, with urine smelling of maple syrup; homocystinuria, with a marfanoid habitus, downward lens dislocation and thrombosis; and alkaptonuria, in which the urine darkens on standing and the cartilage stains black.

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