Part I MCQs (One Best Type)

1. Pakistan Society Of Chemical PathologistsDistance Learning Programme In Chemical Pathology(DLP-2)Lesson No 16Metabolic Bone DiseaseBy Dr Lena JafriMBBS, FCPSConsultant Chemical PathologistAga Khan University Hospital

2. Part IMCQs (One Best Type)

3. Q.1: A 30 year old female complains of backache and generalized weakness. Her biochemical profile was as following : • Calcium: 7.5 mg/dl • Phosphorous: 2.2 mg/dl • Alkaline phosphatase : 107 U/l She is most likely suffering from: • Hypomagnesaemia • Hypoparathyroidism • Osteitisfibrosacystica • Paget’s Disease • Vitamin D deficiency e. Vitamin D deficiency

4. Fat soluble hormone • Major forms are Vitamin D2 (ergocalciferol) and D3 (cholecalciferol) • Receptors have been identified for >30 tissues • Function of the immune, reproductive, muscular, skeletal and integumentary system • VDR best characterized on intestine, kidney and bone • Critical for maintenance of bone health Introduction: Vitamin D

5. Stimulates GI absorption of both calcium and phosphate • Stimulates renal reabsorption of both calcium and phosphate • Stimulates bone resorption Role of Calcitriol ↑ serum calcium ↑ serum phosphate Net effect of calcitriol

6. Sources and Metabolism of Vitamin D

7. Daily need of Vitamin D Exposure of arms & legs for 5-30 min (depending on time, season, latitude & skin pigmentation) between 10am-3pm twice/week With longer exposure to UVB rays, an equilibrium is achieved in the skin, and it degrades as fast as it is generated

8. “deficiency” “Insufficiency” “ Normal” ng/ml 10 20 30 40 50 Serum Levels of 25 hydroxy vitamin D Hollick MF. NEJM. 2007; 266-280. Boonen S et al. Osteoporosis Int. 2004;15:511-519. Lips P. Endocr Rev. 2001;22:477-501. Heaney RP. Osteoporosi Int. 2000;11:553-555. Heaney RP. Am J ClinNutr. 2004; 80 (suppl):1706S-1709S. Thomas MK. NEJM. 1998;388:777-781.

9. Rickets and OsteomalaciaSlides courtesy of Dr Mariam Rafiq, QAMC, Bwp

10. Difference between Rickets and Osteomalacia • Rickets :deficient mineralization at the growth plate • Disease of growing bone. • Refer to changes at the growth plate caused by deficient mineralization of bone. • Occurs before the growth plates fuse. • Osteomalacia: impaired mineralization of the bone matrix • Rickets and osteomalacia usually occur together when growth plates are open only osteomalacia occurs after the growth plates have fused.

11. Calcipenic Rickets Vitamin D deficient ( Nutritional) Rickets Vitamin D dependent Rickets • Type I • Type II Phosphopenic Rickets Hereditary • X-linked Hypophosphatemia(vitamin D-Resistant Rickets) • Autosomal Dominant • Autosomal Recessive • Hypophosphatemic rickets with hypercalciuria Acquired disorder • Tumor-induced (or oncogenic) osteomalacia Classification of Rickets

12. Skeletal findings  • Delayed closure of fontanelles • Parietal and frontal bossing • Craniotabes (soft skull bones) • Rachitic rosary • Harrison Grove • Widening of epiphyseal plate • Progressive lateral bowing of the femur and tibia Radiological • Disorganization of the growth plate with cupping, splaying, cortical spurs, stippling. • Cupping of metaphysis • Looser zones/pseudofractures • Osteopenia Clinical Features

14. Q 2.Following is the bone profile of a technologist working in your laboratory who fractured her ankle because of a minor trauma in the laboratory. • Calcium: 7.2 mg/dl • Albumin : 4 g/dl • Phosphorous: 5.47 mg/dl • PTH: 5 pg/ml The lab investigation you would like to request next would be: • 25-hydroxy vitamin D • Alkaline phosphatase • Creatinine • Magnesium • N-Telopeptide d. Magnesium

15. Secretion of PTH is controlled chiefly by serum [Ca2+] through negative feedback • Calcium-sensing receptors located on parathyroid cells are activated when [Ca2+] is elevated • The G-protein coupled calcium receptors bind extracellular calcium • High concentrations of extracellular calcium result in activation of the Gq G-protein coupled cascade through the action of phospholipase C • This hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to liberate intracellular messengers IP3 and diacylglycerol (DAG) Regulation of PTH Secretion

16. These two messengers result in a release of calcium from intracellular stores and a subsequent flux of extracellular calcium into the cytoplasmic space • This results in an intracellular Caconcentration that inhibits the secretion of preformed PTH from storage granules in the gland • In contrast to the mechanism that most secretory cells use, calcium inhibits vesicle fusion and release of PTH. In the parathyroids, magnesium serves this role in stimulus-secretion coupling • Hypomagnesemia inhibits PTH secretion and also causes resistance to PTH, leading to a form of hypoparathyroidism that is reversible • Hypermagnesemia also results in inhibition of PTH secretion Regulation of PTH Secretion

17. Parathyroid Stimulators • Decreased serum [Ca2+] • Mild decreases in serum [Mg2+] • An increase in serum phosphate (increased phosphate causes it to complex with serum calcium, forming calcium phosphate, which reduces stimulation of Ca-sensitive receptors (CaSr) that do not sense calcium phosphate, triggering an increase in PTH) Parathyroid Inhibitors • Increased serum [Ca2+]. • Severe decreases in serum [Mg2+], which also produces symptoms of hypoparathyroidism (such as hypocalcemia) • Calcitriol

18. Ca binds to CaSR extracellular domain • Conformational changes • Interaction with G protein • Gqa activates phospholipase C • Intracellular Ca altered • Hydrolysis of phospatidyl inositol 4 5 bisphosphate • Inositol 3 phosphate inhibits PTH release Regulation of PTH

20. Q. 3: A 52-year-old woman with menopause at 42 years of age was being evaluated by a physician for fracture risk. She fractured her ankle while getting off a rickshaw 8 months previously. DXA scan showed her T score at the hip was -2.3. Her mother had fractured her hip at the age of 63. She dislikes dairy products and smokes occasionally. The most significant risk factor for her fracture risk, independent of her bone mineral density reading is: • Early menopause • Low calcium intake • Osteoporosis in mother • Previous fracture • Smoking d. Previous Fracture

21. A disease characterized by: • low bone mass • microarchitectural deterioration of the bone tissue Leading to: • enhanced bone fragility • increase in fracture risk Osteoporosis

22. Fractures are the most dangerous aspect of osteoporosis • Debilitating acute and chronic pain in the elderly is often attributed to fractures from osteoporosis and can lead to further disability and early mortality • These fractures may also be asymptomatic • The most common osteoporotic fractures are of the wrist, spine, shoulder and hip Fractures

23. Type of pathologic fracture that occurs as result of normal activities, such as a fall from standing height or less. • There are 3 fracture sites said to be typical of fragility fractures: vertebral fractures, fractures of the neck of the femur, and Colles fracture of the wrist. • This definition arises because a normal human being ought to be able to fall from standing height without breaking any bones, and a fracture therefore suggests weakness of the skeleton. Fragility fracture

24. Extremely high risk • Prior osteoporotic fracture (without significant trauma) • Glucocorticosteroid use (prednisone ≥7.5 mg/d or equivalent for ≥6 months) • Solid organ transplant (pre or post, especially in first 2-3 years) High risk • Glucocorticosteroiduse (prednisone ≥5 mg/day or equivalent, for ≥3 months) • Woman age >65 yrs/man age >70 yrs • Postmenopausal woman or older man with one or more of: • Personal history of low impact fracture • Family history of fracture hip, wrist, or spine (first degree relative age ≥50 yrs) • Currently smoking • Rheumatoid arthritis • BMI <20 • Recent weight loss <10% • Multiple risk factors for falling Clinical risk categories for osteoporosis and osteoporotic fractures

25. Hormonal conditions • Hypogonadism • Late menarche (age >15 yrs) • Early menopause (age <45 yrs) • Cushing's syndrome • Hyperparathyroidism (primary or secondary) • Thyrotoxicosis • Gastrointestinal and nutritional factors • Gastrectomy • Low gastric acid • Celiac disease • Bariatric surgery • Inflammatory bowel disease • Pancreatic insufficiency • Heavy alcohol use • Family history of osteoporosis Clinical risk categories for osteoporosis and osteoporotic fracturesModerate risk

26. Q.4:A 47-year-old female is noted to have a serum calcium level of 11.1 mg/dL (2.75 mmol/L) with normal creatinine at the time of a routine annual visit. Investigations on follow-up after 1 month shows: • Calcium: 11 mg/dL • 25 hydroxy vitamin D 63 ng/ml (Ref Value for normal >30 ng/ml) • Parathyroid hormone 125 pg/ml (16-87 pg/ml) The most likely cause of hypercalcemia is: • Humoralhypercalcemia of malignancy • Primary hyperparathyroidism • Secondary hyperparathyroidism • Tertiary hyperparathyroidism • Vitamin D toxicity b. Primary hyperparathyroidism

27. Hyperparathyroidism

28. Increases the release of calcium from bone matrix • Increases calcium reabsorption by the kidney • Increases phosphate excretion • Increases renal production of 1,25-dihydroxyvitamin D3 (calcitriol), which increases intestinal absorption of calcium Effects of high PTH

29. Q. 5: Blood sample of parathyroid hormone (PTH) is transported in ice because its half-life is: • 20 seconds • 30 seconds • 2-4 min • 8-10 min • 15 min c. 2-4 min

30. Linear peptide consisting of 84 amino acids • PTH present in circulation is very heterogeneous • Heterogeneity is the consequence of a complex metabolism that starts in the parathyroid cells and continues in other tissues, mainly in the kidneys and liver • A circulation pool of “PTH peptides,” not only in pathological conditions, but also in normal individuals PTH molecule

31. In the first gen, noncompetitive, “sandwich” type assay, a single polyclonal antibody competes for labeled PTH and the serum forms • In the 2nd-gen assays, immunometric, 2 distinct antibodies (usually monoclonal), directed against different epitopes, bind the PTH forms present in the sample. One of the antibodies is bound to a solid phase, and the other is labeled • In the third gen assays, the recognition is based on other principles (mass spectrometry) in serum samples previously purified PTH assays: Evolution

32. Recognition of PTH by two different antibodies, one carboxyl terminal and the other amino terminal • Widely available • Adapted to most of the automation platforms • Specificity of the amino terminal antibody defines if the assay measures only the bioactive PTH circulating form (including the first amino terminal amino acids) or the “intact” PTH, which includes, besides bioactive PTH, other “long” carboxyl-terminal forms, for example, 7–84-PTH • Assays for “intact” PTH are the most commonly available and the potential advantage of the bioactive PTH assays is still debatable Immunometric assays a breakthrough

33. The native or intact (1-84) PTH has a short half-life, 2-4 minutes • Whereas the carboxy and midmolecule fragments, which are biologically inactive, have half-lives 10- to 20-fold higher. • The high concentrations of biologically inactive fragments have interfered with use of C-terminal or midmolecule assays for evaluation of parathyroid function in patients with impaired renal function. PTH Half Life

34. Intact PTH assays provide a more accurate assessment of parathyroid patients including those with various renal diseases. • ESRD: predominance of the carboxyl terminal ones, are present in great quantities in comparison to the intact 1–84 form PTH Half Life (Cont)

35. Q. 6: A 65-year-old female, complains of nausea, lethargy and bone pains for last 8-10 months. The investigations on admission showed: • Serum Calcium 7.2 mg/dl • Serum Albumin 2.9 g/dL The corrected calcium (mg/dl) is: • 7.56 • 7.81 • 8.08 • 8.29 • 8.37 c. 8.08

36. One can derive a corrected Ca (adjusted Ca) level, to allow for the change in total calcium due to the change in albumin-bound calcium • Gives an estimate of what the total calcium level would be if the albumin were a specified normal value • Each 1 g/dL decrease of albumin will decrease 0.8 mg/dL in measured serum Ca and thus 0.8 must be added to the measured Cato get a corrected Ca value Corrected Calcium

37. Corrected calcium (mg/dL) = measured total Ca (mg/dL) + 0.8 (4.0 - serum albumin [g/dL]), where 4.0 represents the average albumin level in g/dL. • Corrected calcium (mmol/L) = measured total Ca (mmol/L) + 0.02 (40 - serum albumin [g/L]), where 40 represents the average albumin level in g/L Formula for Corrected Calcium

38. Q. 7: A 55-year-old man with recurrent bone pains was evaluated in a bone clinic. Following laboratory investigations were done: • Serum creatinine: 0.5 mg/dl • Serum phosphate: 1.2 mg/dl • Serum calcium: 8.6 mg/dl • Urinary phosphate: 340 mg/dl • Urinary creatinine: 55 mg/dl The TMP- GFR (mg/dl) of this man is: • 0.25 • 0.06 • 0.05 • -0.25 • -1.88 e. -1.88

39. Tubular reabsorption of phosphate (TRP) • Tubular maximal reabsorption rate of phosphate to GFR (TmP/GFR) Formula for TMP/GFR

40. Q. 8: Following is the biochemical bone profile of a 55-year-old man during routine annual checkup. Bone X-ray was normal. He also gave history of recurrent kidney stones since last 3 years. His biochemical results showed: • Serum Na: 141 mmol/L • Serum K: 3.8 mmol/L • Serum Chloride: 111 mmol/L • Blood Ionized Ca: 3.2 mmol/L • Serum P: 0.86 mmol/L • Serum ALP: 80 U/L • Creatinine: 0.9 mg/dl Investigation which would help in confirming your diagnosis in blood is: • 25-hydroxyvitamin D • FGF-23 • Magnesium • N-telopeptide of type I collagen • Parathyroid hormone e. Parathyroid Hormone

41. Q. 9: If the Parathyroid hormone is undetectable which panel of laboratory investigations will help you make the diagnosis? • 25OHD, PTH-rp , ACE • 25OHD, PTH-rp, NTX • Ca, ACE, ALP • Ca, P, 25OHD • P, PTH-rp, ALP e. Ca, P, 25OHD

42. Identify the mystery molecules! Match the picture to the correct option:

43. 10.1.c NTX

44. 10.2. d. Parathyroid hormone

45. 10.3.b. Bisphosphonate

46. 10.4.e. Telopeptidecross links

47. 10.5.a.1, 25 dihydroxy-vitamin D3

48. Part IIShort Answer Questions:

49. Q.11: A 5 year old girl is brought to a paediatrician for not being able to walk properly since one and a half year. Mother explained that her marriage was consanguineous and two of her boys (10 years and 8 years old) had deformed bones and were unable to walk. On examination, there are skeletal deformities of both upper and lower limbs, frontal bossing and pallor. Her extremities have widened wrists and ankles. The laboratory evaluationincluded: Serum • Creatinine 0.5 mg/dl • Calcium 8.4 mg/dl • Phosphorus 1.8 mg/dl • Alkaline phosphatase 1917 U/l • 25OHD 28 ng/ml (Ref Value for normal >30 ng/ml) • PTH 90 pg/ml Urine • Urinary phosphate 740 mg/day

50. X-ray of wrist and legs was requested: The paediatrician wanted to confirm his suspicion and ordered TMP-GFR in this patient.