1. Clinical Biochemistry and Metabolic disease II Dr Vivion Crowley Consultant Chemical Pathologist St James’s Hospital Dublin

2. Biochemical Investigation of a Patient with Suspected Hypocalcaemia?

3. What are the causes of Hypocalcaemia  Low albumin  Artefact  Chronic renal failure  Hypoparathyroidism  PTH resistance -Pseudohypoparathyroidism -Hypomagneaemia  Vitamin D deficiency  Vitamin D resistance states  Medications  Miscellaneous -Pacreatitis -Sepsis -Tumour lysis syndrome

4. What are the clinical features of Hypocalcaemia PNS: numbness, tingling, muscle cramps CNS: Psychiatric symptoms e.g irritability, rarely tetany and seizures Basal ganglia calcification Chvostek’s sign Trousseau’s sign

5. Does the patient have “True HypoCa”  What is the corrected Ca?  Evidence of artefact? - K EDTA contamination  What is the local reference range?

6. What is the Plasma PO4 level? High PO4 Hypoparathyroidism Renal failure Low/Normal PO4 Vitamin D Renal failure – post dialysis/treatment Other causes NB: PO4 levels are affected by many factors and cannot be relied upon alone  Does the patient have Hypomagnesaemia? -Causes PTH resistance -Must correct Mg deficit before improvement in Ca levels

7. Is there a clinically apparent cause of Hypocalcaemia?  Chronic Renal Failure – high PO4, low 1, 25 (OH) Vit D  Pancreatitis – precipitation of Ca and Mg in necrotic adipose tissue  Sepsis – low albumin  Post thyroid or parathyroid surgery – “Hungry bone syndrome”  Tumour lysis syndrome – high PO4 precipitates Ca  Rhabdomyolysis – as for TLS, marked increase in CK  Osteoblastic metastases

8. What about Calcium-reducing medications  Bisphosphonates  Calcitonin  Phosphate  Foscarnet  Pentamidine

9. What is the plasma PTH Level? Low PTH Hypoparathyroidism -Idiopathic – Di George syndrome -Autoimmune -Post-surgical -Hypomagnesaemia High PTH Vitamin D deficiency or resistance (secondary Hyperparathyroidism) Psuedohypoparathyroidism Severe liver disease Chronic renal failure NB: Check Vit D levels in secondary hyperparathyroidism

10. 43 yr old male with renal failure due to analgeisc nephropathy Na 134 K 5.8 Urea 42.2 Creat 650 Ca 1.82 PO4 2.53 Alb 37 ALP 200 PTH 425 What is the cause of his HypoCa? Explain the abnormal findings?

11. 70 yr old male presented with the Hx of Bone pain and malaise Ca 3.4 PO4 1.5 Alb 30 TP 110 ALP 100 What is the corrected Ca level? What further investigations would you consider? The PTH is 10 (9-65), is the HyperCa PTH dependent or independent? What is the likely diagnosis? What is MGUS?

12. Case 2 GM 73 old female Admitted April and July 2008 with –Left sided weakness, slurred speech, confusion –Mild seizures on 2 nd admission –Poor oral intake, weight loss (57.5 kg on 1/5/08 –48.5 kg on 22/07/08) PMH: HTN, Hypercholesterolaemia, OA Medication: Aspirin, Doxazocin, Perindopril Amlodipine, Omeprazole Pravastatin

13. Case 2 cont. Routine bloods: –GluR 5.5 mmol/L –U/Es: urea 20.9…5.4 mmol/L, creatinine 202…66 umol/L (after rehydration), Na 140 mmol/L, K 2.9 mmol/L –Ca 1.24 mmol/L Albumin 28 g/L, PO4 1.16 mmol/L (ref. 0.8-1.4) –LFTs NAD

14. Case 2 cont. Laboratory tests cont. –FBC- Hb 10.3, MCV 77.7, tTG 1.6, iron 2 (ref 14-3) umol/L, transferin sat. 7 (ref 30-40) %, ferritin and folate NAD –TFTs TSH 0.71 mU/L, fT4 23 pmol/L

15. –25OH Vit D 32 nmol/L (>80) –PTH 148.5 pg/mL (ref 15-65)

16. Case 2 cont Mg 0.15( 0.7-1.0) mmol/L,

17. Case 2 cont. Other investigations: –CXR/PFA NAD –OGD/Colonoscopy NAD –AUS: loss of normal cortical medullary differentiation –MRI brain NAD

18. Case 2 cont. Treatment: –iv Magnesium 7 g in total –Vit D2 50 000 iu im –Magnesium Verla 5 g bd ( 5g equiiv. 5 mmol or 121.5 mg magnesium) –One-Alpha 0.5 mcg (alfacalcidol) for 1/52 –Calcichew D3Forte ii od (one tablet= 400 IU Vit D3, 500 mg Ca++carbonate) –Kay-Cee-L (75 mg KCl per 1 mL)

19. 25/422/728/813/11 Ca1.241.722.442.46 Mg0.150.110.89 K2.92.54.44.6 25 OH VitaminD 323541 79 Albumin28324042 PTH14819

20. Biochemical test play an essential role in the management of endocrine disease Screening – TSH in neonatal hypothyrroidism Case finding – e.g. Pentagastrin test in medullary thyroid ca Diagnosis – over or under production of hormones Monitoring response to Rx or recurrence

21. Disorders of Endocrine Glands  Underactivity with under production of normal hormone  Overactivity with over production of normal hormone  Inappropriate production of hormone not normally produced  Ectopic production of hormone by a non-endocrine gland

22. Disorders involving the Pituitary Gland

23. Hypopituitarism Partial or complete deficiency of anterior and/or posterior pituitary hormones Causes Pituitary tumours Parapituitary tumours e.g. craniopharingioma, metasases Radiotherapy Apoplexy (infarction) Infiltration – sarcoidosis, histiocytosis x, haemochromatosis Infections – TB, abscess Trauma- post head injury Genetic syndromes e.g. Kallmann’s syndrome, Laron dwarfism

24. GHChildren – growth delay Adults – GHD FSH/LHDelayed puberty Oligo/amenorrhoea Erecile dysfunction + testicular atrophy Loss of secondary sex characteristics ACTHhypoadrenalism TSHhypothyroidism ProlactinFailure of lactation AVP (ADH)Diabetes insipidus Hypopituitarism- Clinical Manifestations

25. Hypopituitarism – Biochemical investigation Basal hormone levels -LH, FSH, Testo / E2 -TSH and T4 -9am cortisol -Prolactin (PRL) -IGF-1 (marker of GH action) Dynamic function tests -Insulin tolerance test (ITT) – for Cortisol and GH response -GnRH test for LH, FSH reserve -TRH test for TSH reserve (rarely used now) Also need to consider imaging pituitary gland – MRI, CT

26. Acromegaly - Excess GH secretion Causes Pituitary tumour -Macroadeneoma >10mm -Microadenoma <10mm Clinical presentaion  Pituitary gigantism in childhood  Acromegalic symptoms -Increased sweating -Headaches -Fatigue/tiredness, joint pains -Change in shoe size, ring size

27. Acromegaly – clinical signs Facial appearances - characteristic Deep voice Macroglossia Enalarged hands/feet Carpal tunnel syndrome

28. Biochemical Investigation of Suspected GH Excess Oral glucose tolerance test (75g load) -in acromegaly there is a failure to suppress GH to < 2 mU/L IGF-1 – usually elevated Check other pituitary hormones ( see in Hypopit section) Imaging of pituitary

29. Cushings’ syndrome - Excess circulating cortisol Causes 1.Exogenous steroids 2.ACTH secreting pituitary tumour 3.Ectopic production of ACTH 4.Adrenal tumour Clinical Features -Facial appearance – moon facies, plethoric complexion, acne -Weigt gain – central obesity, “buffalo hump” -Thin skin, easy bruising, -Proximal muscle weakness -Mood disturbance -Menstrual disturbance, hirsutism -Hypertension

30. Cushing’s Syndrome

31. Investigation of suspected Cushing’s syndrome Does the patient have Cushing’ syndrome? What is causing the patient’s Cushing’ syndrome?

32. Does the Patient have Cushing’s syndrome? Out-patient tests Overnight dexamethasone suppression (1mg at midnight) Normal = 9am cortisol < 50nmol/l 24 hour urinary free cortisol In-patient tests Midnight cortisol – looking for loss of cicardian rhythm Low dose dexamethasone suppression test (0.5mg qds for 48hour) Normal = 48hr cortsiol < 50 nmol/l

33. What is the underlying cause of Cushing’s syndrome? Plasma ACTH level  ACTH-dependent if non-suppressed or elevated - Pituitary - Ectopic  ACTH independent if suppressed -Adrenal To differentiate Pitutary ad ectopic causes  High dose dexamethasosne suppression (2mg qds x 48hr) -Pituitary suppresses to < 50% basal value  CRH test -exagerated cortisol and ACTH response in pituiray Cushing’s Inferior petrosal sinus sampling + pituitary imaging

34. Hyperprolactinaemia ConditionExample PhysiologicalPregnacy, lactation Idiopathic Stressvenepuncture DrugsDopamine agonistse.g Phenothiazines Chronic liver/renal diseaseCirrhosis, CRF Hypothalamic/pituitary disorders Micro/macroadneoma Stalk compression syndrome Primary hypothyroidism PCOS

35. Hyperprolactinaemia – clinical presentation Females -Oligo/amenorrhoea -Galatorrhoea -Infertility -Reduced libido -Symptoms relating to pituitary tumour Males -Symptoms relating to pituitary tumour e.g. headache, visual disturbance# -Less frequently, reduced libido, infertility, galatorrhoea

36. Biochemical investigation of Hyperprolactinaemia  Plasma Prolactin -At least two confirmed elvations in plasma prolactin -Must screen for Macroprolactinaemia ( a high mol wt form of circulating prolactin) -Macroprolactinaemia is not clinicall significant -Prolactin levels > 5000mU/L are indicative of a pituitary adenoma  Check routine bloods -renal and liver function -TFTs -Pregnancy test (hCG) -FSH/LH, E2  Imaging of pituitary gland if indicated

37. What are the commonly measured TFTs?  Total T4 – includes protein bound and Free Thyroxine -Free Thyroxine is the active hormone -TBG is the main protein binding Thyrxoine -Increased TBG s seen in pregnancy  Free T4 (FT4)  TSH – reflects the pituitary response to FT4 level  Total T3 – useful in the diagnosis of T3 toxicosis - Normal T4 and suppressed TSH

38. Patterns of Thyroid Function Tests

39. Hypoglycaemia Definition: plasma glucose < 2.8mmol/l (blood glucose < 2.2mmol/l) Clinically presents with - adrenergic features, neuroglycopaenia “Whipple’s triad” - Symptoms & signs of hypoglycaemia Plasma glucose < 2.8mmol/l Relief of symptoms by glucose intake (infusion/oral)

40. Hypoglycaemia Causes: Drug therapy - Insulin, Sulphonylurea,  -blockers, Quinine Factitious - Insulin, sulphonylureas (healthcare workers) Fasting Hypoglycaemia  Insulinoma  Hepatic failure - gluconeogenesis  Sepsis, Cardiac failure  Hypopituitarism, Addison’s disease  Tumour-related hypoglycaemia - mesenchymal tumours e.g. fibrosarcoma etc. ? Ectopic IGF II by tumour cells  Autoantibodies - Insulin, Insulin receptor

41. Hypoglycaemia Reactive Hypoglycaemia Idipopathic Early diabetes Post-gastric surgery Investigations: Ensure that hypoglycaemia is documanted by laboratory blood/plasma glucose Determination on a sample collected into a fluoride tube 5hour OGTT - hypoglycaemia may occur between 2-5 hours after glucose load Definitive investigation for fasting Hypoglycaemia: Supervised - 72 hour prolonged fast If pt develops neuroglycopaenic symptoms then measure Plasma Glucose, Insulin, C-pepetide Other routine invsetigations: U/E, LFTs, ? Endocrine

42. Polyuria Urine output > 3 litres/day (explain the difference between polyuria and urinary frequency) Confirm polyuria - 24hr urine collection Causes of polyuria Drugs - diuretics, lithium Diabetes mellitus - fasting ± random glucose, OGTT Chronic renal failure - plasma urea & creatinine, Creatinine clearance Hypokalaemia Hypercalcaemia

43. Polyuria In clinical practice the most common reason for doing a water deprivation test is to differetiate between Psychogenic polydipsia Diabetes Insipidus - central AVP (ADH) deficiency - nephrogenic AVP action on renal tubule Water Deprivation test ± vasopressin administration

44. Addison’s Disease Primary adrenal insufficiency - differentiate from secondary i.e. ACTH deficiency - tertiary :CRH suppression by exogenous steroids Clinically  Weakness, fatigue, anorexia, wt loss, postural hypotension, Coma  Hyperpigmentation - elbows, knees, buccal mucosa, recent scars (ACTH levels) Causes Autoimmune - ? Polyglandular autoimune syndromes Infections - TB, fungal infections, HIV Metastatic disease - lung, Breast, colon (bilateral disease) Medications - ketoconazole Waterhouse Freidrickson - bilateral adreanl haemorrahage (sepsis e.g menigococcus)

45. Biochemical abnormalities in Addison’s Disease Hyponatraemia Hyperkalaemia Pre-renal failure plasma urea & creatinine ? Hypercalcaemia Abnormal TFTs - TSH, T4 (hypothyroid picture) - May be a transient picture NB: treat hypoadrenalism before giving L-T4 Acute presentation: Take blood sample for plasma Cortisol and ACTH levels before steroid administration Plasma Cortisol Plasma ACTH NB: Dexamethasone does not interfere with cortisol assay

46. Biochemical Diagnosis of Addison’s disease Chronic Presentation  Short synacthen test -does not differentiate between primary and secondary -Synacthen 250 μg IM -0, 30 and 60 min plasma cortisol -30 min Plamsa Cortisol > 550 nmol/L  Long synacthen test  Adrenal antibodies

47. Biochemical paraneoplastic syndromes  Tumours of specific endocine glands e.g. insulinoma, pituitary etc.  Hypercalcaemia of malignancy  Syndrome of inappropriate antidiuresis (SIAD) - hyponatraemia  Cushing’s syndrome  Tumour-induced hypoglycamia  Tumour-induced osteomalacia - mesenchymal tumours (hemangiopericytoma) Normal Ca, PO4, 1,25 (OH)2 Vit D ??Renal phosphate wasting  Carcinoid syndrome - facial flushing, diarrhoea, brochospasm Laboratory diagnosis - urinary 5 hydroxyindole actetic acid (5HIAA)

48. Case 1: Hx  35 yr old male  Hx EBV infection 8 weeks previously  No medical or family hx of note otherwise  Not on regular medication  C/O vague/nonspecific symtoms, fatigue  GP performed “routine blood tests” including TFTs

49. Case 1: TFTs  TT4= 223 (63-142)  TSH= 4.87  Repeated 2-3 weeks later  TT4 = 171  TSH = 2.89

50. Case 1: TFTs (cont)  FT4 = 51.3 (9-24)  T3 = 3.5 (0.8-2.5)  Biochemical Hyperthyroidism  No clinical features of thyrotoxicosis  No biochemical evidence of heterophile ab interference  No family hx of thyroid dx

51. Case 1: Differential Dx  TSHoma  Thyroid hormone resistance (RTH) - Refetoff’s syndrome  FDH – rare  Heterophile abs  MRI scan of pituitary – Macroadenoma - TSHoma

52. Case 2: History  Phone call from a GP regarding TFTs:  54 yr old male – vague hx fatigue  T4 = 40 (69-141)  TSH = 1.53  Sample recovered and tested for  FT4 = 5.7 (9-24)  TBG = 25 (13-24)  A large clot was noted in serum  R/O artefact – repeat investigation with

53. Case 2: Repeat TFTs TT4 = 44 FT4 = 4.9 TSH= 1.2 Cortisol = 130 (random am) Testo = 10.5 (8.7 –33.0) LH = 2.4 FSH = 6.3 GH < 1.0 IGF-1 < 25 PRL = 419 (70-413) Central Hypothyroidism – Referred to endocrine service SJH

54. Case 2: Endocrine assessment  Stimulation testing (ITT) -Achieved hypoglcaemia -GH deficient -ACTH/Cortisol deficient -Centrally hypothyroid MRI scan – empty sella ? cause

55. Case 3: Background  23 yr old male  Hx Migraine and fatigue  GP performed TFTs  TT4 = 43  TSH = 2.89  Biochem Registrar signing out - ? Hypothyroid - Put on some additional test  FT4 = 5.8 (9-24)

56. Case 3: Endocrine tests  More additional tests after discussion with GP Cortisol = 97 (244-727) but NB diurnal variation FSH = 2.7 (1.0-10.5) LH = 2.2 (1.0 –6.0) Testo = 1.3 (8.7-33) Prolactin = 4490 (70-413) Post fractionation Prolactin (PFP) = 3960

57. Case 3: Diagnosis  Biochemical Dx: -Hypothyoid -Hypogonadal -Hypocortisolaemic -Hyperprolactinaemic  Hypopituitarism  MRI scan – Macroadenoma - ? Macroprolactinoma

58. Case 4: background  43 yr old female  Hx Spina Bifida (wheel chair bound)  Currently in Cheshire Home  4/52+ hx malaise  Admitted to St Elsewhere  Atonic Bladder  Renal impairement  GP to monitor U/Es

59. Case 4: 5/56/512/5 Na165162 K3.63.0 Urea14.26.9 Crea167133 POsmo352 UOsmo410222 Gluc13.69.7* * fasting Result brought to attention of Consultnat Chem Path Hypernatraemia ? Cause

60. Case 4: Dx? Results suggestive of -DI -DM Paired urine and plasma osmolality are very useful in directing management of -hypernatraemia -hyponatraemia

61. 24 yr old male Unwell – Hx of admissions with hypoNa Na 121 K 5.5 Urea 9.3 Crea 118 T4 83 TSH 4.97 Cortisol 52 ACTH 1122