Clinical Chemistry II: Electrolytes

Presented by

Mark Wilson Lozada, RMT

UERMMMCI – College of Allied Health Professions
2

Outline
General Physiology and Laboratory Analysis of Electrolytes
Electrolyte – Chemistry and Biological Functions
Overview of Electrolyte Determination in the Clinical Laboratory
Specimen Collection and Processing
Instrumentation and Analytical Methodologies
Trends seen in some diseases associated with electrolytes
Anion Gap
Water Distribution & Physiology
Total Body Water
Water Homeostasis
Plasma osmolality
Sodium
Potassium
Chloride
Bone and Mineral Metabolism
Bone and Mineral Metabolism
Calcium
Phosphate
Magnesium
General Physiology &
Laboratory Analysis
4

Electrolytes

Particles or compounds that carry an electrical

charge
A substance which dissociates into ions when
dissolved in water
cation (+ charge) or an anion (- charge)
Major Electrolytes
Na+, K+, Cl-, HCO3-
5

Electrolytes

Intracellular Extracellular
Cations Cations
K+ Na+
Mg2+ Ca2+
Anions Anions
PO43- Cl-
6

Electrolytes : Biological Functions

Volume and Osmotic regulation

Sodium, Potassium, Chloride
Myocardial rhythm and contractility
Potassium, Magnesium, Calcium
Enzyme cofactors
Magnesium, Calcium, Chloride*
Regulation of Ion pumps
Magnesium, Calcium, Potassium
7

Electrolytes : Biological Functions

Acid-Base Homeostasis
Potassium, Chloride, Bicarbonate
Blood Coagulation
Calcium, Magnesium
Neurotransmitter Excitability
Potassium, Calcium, Magnesium
ATP Usage and Production
Magnesium, Phosphates
8

Electrolyte Determination

Electrolyte Profile
Na+, K+, Cl-, HCO3-
Serum or Urinary Calcium
Total Calcium
Ionized Calcium (Serum only)
Serum or Urinary Magnesium
Serum or Urinary Phosphates
Sweat Chloride
9

Electrolyte Determination

Common Diseases associated with Electrolyte testing

Dehydration
Diarrhea
Vomiting
Burns
Kidney Disease
High Blood Pressure (Diuretic Use)
Acidosis / Alkalosis
10

Specimen Collection & Processing

Serum or Plasma – usual specimen
Difference in value measured only significant in K+ and PO43-
K+ : Higher in serum by 8.4%
PO43- : Higher in serum by 7.0%
Lithium Heparin anticoagulant for plasma or whole blood
Hemolysis : increased K+,Mg2+, PO43- ; decreased Na+, Cl-
Lipemia : decreased electrolytes measured by indirect ISE
Samples may be stored at 4°C except for :
Whole Blood K+ – increases by 2 mmol/L after 4 hrs
Separate serum (and plasma) from red cells immediately
(within 1 hr.)
K+ : increases by 0.2 mmol/L after 1.5 hrs
Other electrolytes increased : Mg2+, PO43
Prolonged Torniquet application, clenching of fist
K+ : increases up to at least 2 mmol/L
Ca2+ : increases total Ca by 0.5-1 mg/dL ; iCa (clenching of fist)
Lactate : do not use torniquet
11

Electrolyte Exclusion Effect

12

Na+/K+ ATPase Pumps

13

Specimen Collection & Processing

Urine
24-hr urine – preferred specimen
No Preservative : Na+, K+, Cl-
Acidified Urine : Ca2+, Mg2+, Phosphates
Sweat
For chloride testing ; at least 100 mg
Two-step collection procedure
Pilocarpine Iontophoresis – stimulation for 5 mins
Sweat Collection by using either :
 Filter Paper or Gauze (Gibson-Cooke Method)
 Plastic Microbore Tubing
14

Instrumentation & Analysis

Potentiometry by Ion-Selective
Membrane Electrodes
measurement of electrical potential difference
between two electrodes
Direct ISE
Specimen brought directly in contact
with electrode
Common in blood gas analyzers, stand-
alone electrolyte analyzers and POCT
Devices
Not affected by Electrolyte Exclusion
Effect
Indirect ISE
Specimen is diluted with ionic buffer
prior to measurement
Common among automated chemistry
analyzers
Affected by EEE
15

ISE Ionophores for Electrolyte Determination

Potassium
Valinomycin
Bis(benzyl-15-crown-5)-heptanedoate
Sodium
Methylmonensin
ETH 157 and 227
Glass Aluminum silicate
Chloride
Tri-n-octylpropylammonium chloride
Tributylin
Mercuracarborand-3 (MC3) – liquid membrane
Calcium
ETH 1001
Calcimycin
Liquid Membrane : 2-ethylhexyl phosphoric acid in dioctyl phenyl phosphonate
16

Electrolyte Analyzer (Roche® 9180 Electrolyte Analyzer) (Direct ISE)

17

Instrumentation & Analysis

Amperometry
Voltametric technique
involves application of a constant reducing or oxidizing potential to an indicator
(working) electrode and the subsequent measurement of the resulting steady-
state current
Coulometry
measures the electrical charge passing between two electrodes in an
electrochemical cell
Amount of charge (coulomb) passing between the electrodes is directly
proportional to
oxidation or reduction of an electroactive substance at one of the electrodes
Absolute method; gold standard for serum/plasma chloride determination
18

Instrumentation & Analysis

Amperostatic Coulometry (Amperometric-Coulometric Titration)
Employed in Chloride determination; used by Cotlove chloridometer
Anode (+) : Ag wire | Cathode (–) : Pt wire
At a constant applied current, the number of coulombs passed between the anode and
the cathode is directly proportional to time (coulombs = amperes × seconds)
Coulometry Phase
▹ Anodic oxidation reaction to generate silver ions when an electric potential is applied
▹ Ag  Ag+ + e-
▹ Silver ions enter the solution at a rate proportional to electric current
▹ Ag+ + Cl-  AgCl
▹ Amperometry Phase
▹ Endpoint detection : depleted Cl-, excess Ag+
▹ Excess Ag+ is sensed amperometrically at a second Ag electrode, which is
polarized at negative potential
▹ excess Ag+ is reduced to Ag, producing a current. When this current exceeds a
certain value, the titration is stopped
▹ absolute number of Cl− ions present in the sample is calculated from the time during
which titration with Ag+ was in progress
19

Amperometric-Coulometric Titration
20

Trends seen in some diseases

Renal Failure
Increased K+, Mg2+, PO43-, Ca2+
Decreased Na+
Hyperaldosteronism
Increased Na+, Cl-
Decreased K+, Mg2+
Hyperparathyroidism
Increased Ca2+
Decreased PO43-
Alkalosis
Decreased K+, Ca2+
21

Anion Gap

Difference between unmeasured anions and unmeasured cations

Equations to calculate AG
AG = Na+ - (Cl- + HCO3-) | Reference Range : 7-16 mmol/L
AG = (Na+ + K+) – (Cl- + HCO3-) | Reference Range : 10-20 mmol/L
Elevated AG
Uremia/Renal Failure
Ketoacidosis
Methanol, Ethanol, Ethylene glycol, or salicylate poisoning
Lactic Acidosis
Hypernatremia
Instrument error
Relegated AG (rare)
Hypoalbuminemia
Severe hypercalcemia
Water Distribution and
Physiology
23

Total Body Water

75% Interstitial

1/3 ECF
25% Intravascular

2/3 ICF
24

Water Homeostasis
Electrolytes involved : Na+, K+, Cl-
Law of Electroneutrality
the sum of positive and negative charges within the water must balance
to zero (ECF charge = ICF Charge)
Na+ = primary ECF cation
K+ = primary ICF cation ; countercurrent ion of sodium
Cl- = primary ECF anion ; counterion of sodium
PO43- = primary ICF anion
Gibbs-Donan Eqilibrium
If solutions on two sides of a membrane contain different [ions] that
cannot freely move through the membrane (e.g., proteins), distribution of
diffusible ions at the steady state will be unequal but
the sum of [ions] in one compartment is equal to the other
compartment
25

Gibbs-Donnan Equilibrium

• Proteins attract small diffusible cations to

maintain electroneutrality.
• Influx of cations increases osmotic
concentration of the intracellular
compartment.
• To counteract the influx of water, cells
utilize the Na+/K+-ATPase pump to limit
the number of osmotically active cations.
26

Volume Regulation
Salt content
Chief determinant of extracellular volume
Effective Vascular Volume
Chief determinant of how much ECF is retained
Arginine Vasopressin Peptide (AVP)
(Antidiuretic Hormone)
Promotes water retention
Baroreceptors in carotid sinus, atria of heart,
and aortic arch send neural signals to CNS
resulting in an increase or a decrease in AVP
secretion
Aldosterone
Promotes sodium retention
27

Volume Regulation
Natriuretic Peptides : promotes sodium excretion
Atrial Natriuretic Peptide – produced by the atrium
Brain Natriuretic Peptide – produced by the heart ventricles
Marker of heart failure
Urodilatin
Most important natriuretic peptide for renal excretion of sodium

Nonrenal Loss of Water

Skin : 30 mL per 100 calories of heat
Insensible perspiration – water loss from skin without sweat
Sweating
Respiration : 13 mL per 100 calories
GI Tract : 100 mL per day in feces
Fluid lost is isotonic or slightly hypotonic
28

Water Homeostasis
29

Plasma Osmolality

Concentration of dissolved solutes in the body

Measures electrolyte-water balance
mOsmol/kg
= 1.86 [Na+ (mmol/L)] + Glucose (mmol/L) + Urea (mmol/L) + 9
𝒎𝒈 𝒎𝒈
𝑮𝒍𝒖𝒄𝒐𝒔𝒆 ( ) 𝑼𝒓𝒆𝒂 ( )
or 1.86 [Na+ (mmol/L)] + 𝒅𝑳
+ 𝒅𝑳
+9
𝟏𝟖 𝟐.𝟖

Reference interval : 275 – 300 mOsmol/kg

Measured by freezing point osmometer
30

Electrolyte Regulation in Renal Tubules

Sodium reabsorption can occur through three
mechanisms:
70% of Na in filtrate reabsorbed in PT by iso-osmotic
reabsorption – limited by Cl- availability
Na+ reabsorbed in exchange for H+
Aldosterone : Na+ in exchange for K+

K+ reabsorption occur in two mechanisms

Active reabsorption in PT
Exchange with Na+ stimulated by Aldosterone. H+
competes with K+.

Cl- reabsorbed by passive transport in PT along

concentration gradient created by Na+.
31

Electrolyte Regulation in Renal Tubules

Phosphate reabsorption is inhibited by PTH and

increased by 1,25-[OH]2-D3. Excretion of PO4− is
stimulated by calcitonin.
Ca2+ is reabsorbed under the influence of PTH and
1,25-[OH]2-D3. Calcitonin stimulates excretion of
Ca2+.
Mg2+ reabsorption occurs largely in the thick
ascending limb of Henle’s loop.
Bicarbonate is recovered from the glomerular filtrate
and converted to CO2 when H+ is excreted in the
urine.
32

Other Regulatory Processes in the Kidneys

Loop of Henle
Na+-K+-Cl- Cotransporter (NKCC) aids in secondary active transport of
these ions into cells
AVP creates osmotic gradient that enables water reabsorption to be
increased or decreased in response to fluid changes in osmolality.

Collecting Ducts
Under AVP influence ; final adjustment of water excretion
33

Sodium
Major extracellular cation
Major contributor of osmolality
Concentration largely depends on water
Reabsorption is stimulated by
aldosterone
Excretion is stimulated by ANP
Counterbalanced by Cl-
Countercurrent exchange with K+
Na+/K+-ATPase pump: 3Na+ Out, 2K+ In
Reabsorbed in exchange for K+ or H+
Reference Interval
135 mmol/L to 145 mmol/L
34

Hyponatremia
Most common electrolyte disorder
Clinical definition : plasma sodium level < 135 mmol/L
Clinical concern arises when plasma sodium drops to < 130 mmol/L
Clinical Manifestations
Nausea and generalized weakness
Mental confusion (< 120 mmol/L)
Severe mental confusion, seizures (< 105 mmol/L)
Correction must be performed gradually (0.5-2.0 mmol/L/hr)
May cause brain demyelination if too rapid
Classifications
Hypo-osmotic
Iso-osmotic
Hyperosmotic
35

Hypo-osmotic Hyponatremia
May be due to :
Excess loss of Na+ (depletional hyponatremia) – hypovolemic (most common)
Increased ECF volume (dilutional hyponatremia) - hypervolemic
Depletional hyponatremia
Extrarenal Loss : Urine Na+ < 10 mmol/L
GI Loss : Vomiting, Diarrhea
Skin loss : Burns, Sweating
Renal Loss ; Urine Na+ > 20 mmol/L
Diuretics use
Mineralocorticoid deficiency
Metabolic alkalosis : increased HCO3- excretion is accompanied by Na+
Proximal RTA : defective HCO3- reabsorption, Na+ lost along with HCO3-
Ketonuria : lost alongside Ketones
Carbonic anhydrase inhibitors
Salt-losing renal diseases
Polycystic kidney, Chronic interstitial nephritis, obstruction
36

Hypo-osmotic Hyponatremia
High ECF ;
baroreceptors
Dilutional hyponatremia Low BV
High Urinary Na+
Renal Failure
Low Urinary Na+
Congestive Heart Failure
Cirrhosis with ascites
Nephrotic syndrome Intravascular fluid Increase aldosterone
moves to tissues and vasopressin

Congestive Heart Increased venous

Failure pressure

Cirrhosis, Nephrotic Decreased Decreased colloid Increased Sodium and

syndrome Proteins Water retention, ECF
osmotic pressure
further increased, sodium
diluted
37

Hypo-osmotic Hyponatremia
Euvolemic Hypo-osmotic Hyponatremia
Syndrome of inappropriate ADH secretion
(SIADH)
Malignant, Inappropriate vasopressin release
leading to excessive water retention
Primary polydipsia (psychiatric or hypothalamic
as seen in sarcoidosis)
Hypothyroidism
Impaired free water excretion
Hypoadrenalism
Low aldosterone  high cortisol-releasing
hormone
CRH stimulates vasopressin
38

Iso-osmotic Hyponatremia

Causes
Pseudohyponatremia
Electrolyte exclusion effect
Hemolysis
Increased non-sodium cations
Lithium excess
Increased gamma-globulins
Severe hyperkalemia
Severe hypermagnesemia
Severe hypercalcemia
39

Hyperosmotic Hyponatremia

Causes
Severe hyperglycemia
For every 100 mg/dL increase in glucose level that is above 100 mg/dL, Na+
decreases by 1.6-2.0 mmol/L
Mannitol and glycine infusion
Uremia
40

Hypernatremia
Clinical definition : plasma Na+ > 150 mmol/L
Always hyperosmolar
Symptoms : Ataxia, tremors, irritability, confusion, coma
Acute Hypernatremia : symptoms at 160 mmol/L
Chronic Hypernatremia : symptoms may not occur until Na+ exceeds 175 mmol/L
Rapid correction may induce to cerebral edema and death
Classifications
Hypovolemic Hypernatremia
Euvolemic Hypernatremia
Hypervolemic Hypernatremia
41

Hypovolemic Hypernatremia
Dehydration with or without sodium excess
Extrarenal loss of water coupled with failure to replace water
Urine Na+ <10 mmol/L, Hypertonic Urine
Diarrhea
Burns
Fever
Excessive sweating
Renal loss
Osmotic diuresis
Hyperglycemic nonketotic syndrome – common in T1DM patients with glucose
> 600 mg/dL
42

Euvolemic Hypernatremia
Often a prelude to hypovolemic hypernatremia
Causes
Diabetes insipidus : Hypotonic urine
Central DI : decreased or absent vasopressin
Head trauma, hypophysectomy, pituitary tumor, granulomatous disease
Nephrogenic DI- renal resistance to vasopressin
Excessive drug use: lithium, demeclocycline, amphotericin,
propoxyphene
Hypercalcemia
Hypokalemia
Sickle cell anemia
Sjogren syndrome
Mutated vasopressin receptor
Increased insensible loss of water : Hypertonic urine
43

Hypervolemic Hypernatremia
Water excess with a large Na+ excess
Causes
Hyperaldosteronism
Cushing syndrome (Primary Hypercortisolism)
Hypertonic IV fluid therapy
44

Sodium Determination
Direct/Indirect ISE – Glass aluminum silicate
Most widely used
AAS
Flame-emission spectrophotometry – obsolete
Lithium as internal standard
Spectrophotometry
Kinetic Assay using β-galactosidase
Sodium activates the enzyme
Substrate : o-nitrophenyl-β-D-galactopyranoside
Product : o-nitrophenol
Measured at 420 nm
45

Potassium
Major intracellular cation; Excretion is promoted by aldosterone
Countercurrent ion of Na+
Counter-exchange ion of H+
For every 0.1 unit drop in pH, plasma K+ increases by 0.2-0.5 mmol/L
Intracellular concentration is 20x greater than plasma concentration
Entry to skeletal muscle cells & hepatocytes promoted by insulin
Cellular entry promoted by catecholamines, inhibited by propanolol
Exercise increases plasma K+ by 0.3 to 1.2 mmol/L
May increase up to 2-3 mmol/L if exhaustive exercise
Reversed after several minutes of rest
Reference Interval – narrow, any deviation is life threatening
Serum : 3.5 – 5.1 mmol/L
Plasma : 3.5 – 4.5 mmol/L (M) ; 3.4 – 4.4 mmol/L (F)
46

Hypokalemia
Plasma K+ < 3.5 mmol/L
Signs and Symptoms : muscle weakness, irritability, paralysis
< 3 mmol/L : neuromuscular symptoms ; critical intracellular depletion
Lower concentrations : tachycardia, flattened T-waves on ECG
May lead to cardiac arrest
Classification
K+ Redistribution-related Hypokalemia – transient
Insulin response
Alkalosis
Catecholamine or B-adrenergic excess
Leukocytosis (pseudohypokalemia)
Hypothermia
Hypokalemic periodic paralysis
True K+ deficit Hypokalemia
Renal cause
Nonrenal cause
47

Hypokalemia
True K+ deficit Hypokalemia with > 25 mmol/day Urine K+
Renal loss
Acute Tubular Necrosis – diuretic phase
Amphotericin B toxicity
Hypomagnesemia
Metabolic Alkalosis : H+ reabsorbed in exchange for K+
w/ Urine Cl- >10 mmol/d
Mineralocorticoid excess
Cushing syndrome
w/ Urine Cl- <10 mmol/d
Diuretics
Vomiting
Penicillin
Renal Tubular Acidosis
Bartter syndrome
Gitelman syndrome
48

Hypokalemia
True K+ deficit Hypokalemia with < 25 mmol/day Urine K+
Extrarenal loss
Diarrhea
Fistula
Excessive sweating
Decreased dietary intake
Starvation
49

Hyperkalemia
Plasma K+ > 5 mmol/L
Signs and symptoms
mental confusion, weakness, tingling, flaccid paralysis of extremities
>6.5 mmol/L : bradycardia, prolonged PR & QRS intervals, peaked T-waves on ECG
>7.0 mmol/L – peripheral vascular collapse, cardiac arrest
>10.0 mmol/L – absolute fatality
Result of (singly or in combination) :
Redistribution
Increased intake
Increased retention
50

Hyperkalemia
Redistributive Hyperkalemia
Metabolic acidosis
Dehydration
Massive tissue hypoxia
Insulin deficiency
Rhabdomyolysis
Severe burns
Tumor lysis syndrome
Epilepticus
Fluoride poisoning
Iatrogenic
Digitalis/Digoxin toxicity
B-adrenergic blockers
Chemotherapy agents (mitomycin-C, methotrexate, platinum compounds)
51

Hyperkalemia
Retentive Hyperkalemia
Decreased K+ excretion
Mineralocorticoid deficiency
Addison’s disease
Hyporeninemic hypoaldosteronism
ACE inhibitors
Primary renal tubule defect
Obstructive nephropathy
Renal transplant
Sickle cell disease
Systemic lupus erythematosus
K+ sparing diuretics (triamterene, amiloride, spironolactone)
Immunosuppressants (Tacrolimus, Cyclosporine)
Oliguria plus increased K+ load
Massive blood transfusion
Hemolysis
Tissue necrosis
High-dose penicillin
52

Hyperkalemia
Pseudohyperkalemia
Hemolysis
Thrombocytosis
Leukocytosis plus delayed transport or serum-cell separation
Fist pumping during venipuncture
53

Potassium Determination
Direct/Indirect ISE : valinomycin gel
Most widely used
AAS
FES – obsolete
Spectrophotometry
Pyruvate Kinase-Lactate dehydrogenase
Based on K+ activation of pyruvate
Substrate : Phosphoenolpyruvate
Measures disappearance of NADH
Measure decrease in absorbance at 380 nm
54

Chloride
Major extracellular anion
Limiting ion for the reabsorption of Na+
Chief counterion of Na+
Counter-exchange ion of HCO3-
(chloride shift)
Cornerstone for management of acid-base
imbalances
Only anion that has an enzyme-cofactor
function
Amylase
Reference Interval
98 – 107 mmol/L
55

Hypochloremia
Plasma chloride < 98 mmol/L
Causes
Hyponatremia and those associated with it
Prolonged vomiting
Metabolic Alkalosis
Compensated Respiratory Acidosis (normal sodium)
Increased bicarbonate = increased chloride shift
56

Hyperchloremia
Plasma chloride > 107 mmol/L
Causes
Hypernatremia and those associated with it
Metabolic Acidosis
Salicylate intoxication
Compensated Respiratory Alkalosis (normal sodium)
Decreased bicarbonate = decreased chloride shift
57

Chloride Determination
Direct / Indirect ISE : tri-n-octylpropylammonium chloride
Most commonly used
Mercurimetric Titration (Schales and Schales Method)
Indicator : Diphenylcarbazone
End-product : HgCl2 (blue-violet)
Amperostatic Coulometry
Replaced by ISE ; still used for sweat chloride analysis
Spectrophotometry
Whitehorn Titration (Mercuric thiocyanate) – formation of red complex
Ferric Perchlorate : formation of yellow Iron(III)-chloride complex
Iwasaki-Utsumi-Ozawa : Formation of red-orange complex Fe(CNS)2+
Common interferants in all methods
Thiocyanates, other Halide ions
Bone and Mineral
Metabolism
59

Bone and Mineral Metabolism

Bone is a tissue that undergoes continuous remodeling.
Bone remodeling is tightly linked with Ca2+ Mg2+, and PO43- metabolism.
Bone formation – synthesis ; uptake of minerals
Stimulated by calcitriol and calcitonin
Osteoblastic activity
Bone resorption – deconstruction ; release of minerals
Stimulated by PTH
Osteoclastic activity
Hormonal regulation
PTH : sensitive to plasma calcium levels ; secreted by chief cells
Induces bone resorption for Ca2+ and HPO4-
increases renal reabsorption of Ca2+ in exchange for PO43-
Calcitriol : activated form of vitamin D ; activated by PTH in kidneys
Increases intestinal absorption and renal reabsorption of Ca2+ and PO43-
60

Bone and Mineral Metabolism

61

Calcium
Most abundant cation in the body
99% in skeleton
1% in ECF and Soft Tissues
Distribution in plasma
50% - ionized; biologically active form
40% - protein-bound (80% albumin)
10% - complexed
Hormone Regulators
PTH : hypercalcemic hormone
Calcitonin : hypocalcemic hormone; secreted by parafollicular C cells
Reference intervals
Total Ca2+ : 2.15 – 2.50 mmol/L (8.6 – 10.0 mg/dL)
Serum iCa2+: 1.16 – 1.32 mmol/L (4.6 – 5.3 mg/dL)
Plasma iCa2+ : 1.03 – 1.23 mmol/L (4.1 – 4.9 mg/dL)
62

Hypocalcemia
Total plasma Ca2+ < 2.15 mmol/L
Increases neuromuscular excitability
Signs and symptoms (usually when Total Ca2+ drops below 1.88 mmol/L)
Parasthesia : circumoral, peripheral, perianal
Neuromuscular irritability : weakness, twitching, cramps, tetany
Laryngospasm and bronchospasm in severe cases
Fatigue
Cataracts
Cardiac : hypotension, arrhythmia as, ECG abnormalities, CHF
CNS abnormalities
Depression
Alerted mental status
Seizures
Coma
63

Hypocalcemia
Artefactual
Hypoalbuminemia – most common cause
1 g/dL albumin binds 0.02 mmol/L Ca2+
Normal ionized calcium
Usage of EDTA or Citrate in venipuncture
Gadolinium salts
Excessive or rapid infusion of IV Fluids
64

Hypocalcemia
Diseases
Chronic Kidney Disease
Hyperphosphatemia, PTH resistance, Decreased Calcitriol
Hypoparathyroidism
Pseudohypoparathyroidism
Activating CaSR mutations
Osteoblastic metastasis
Hungry Bone syndrome – healing phase
Post-parathyroidectomy, post-thyroidectomy, treatment of hematologic
malignancies
65

Hypocalcemia
Calcium deposition in Tissues
Crush injury
Rhabdomyolysis
Hemorrhagic and edematous Pancreatitis
Tumor lysis syndrome
Vitamin D Deficiency or Resistance
Lack of sunlight exposure
Nutritional deficiency
Malabsorption
Gastric bypass surgery
End-stage liver disease
CKD
Vit. D-dependent rickets types 1 and 2
66

Hypocalcemia
Toxicities
Drugs
Biphosphonates
Deosumab
Imatinib
Proton pump inhibitors
Phosphate enemas
Foscarnet
Aluminum toxicity
Iron overload
Copper toxicity
67

Hypercalcemia
Total Plasma Ca2+ > 2.50 mmol/L
Signs and symptoms
Gastrointestinal Renal
nausea / Vomiting Polyuria
Abdominal pain Dehydration
hyperacidity Prerenal uremia
Peptic ulcers Stone formation
Constipation Tissue Calcification
Pancreatitis Nephrocalcinosis
Neurologic Vascular
Weakness Cardiac
Fatigue Cornea (band keratopathy)
Decreased muscle contaction Gastric mucosa
Depression Cardiac
Obtundation Prolonged Q-T interval in ECG
coma Decreased muscle contraction
68

Hypercalcemia
Primary Hyperparathyroidism
Adenoma, Hyperplasia, Carcinoma
Familial
Familial Benign Hypercalcemic Hypocalciuria
PTH normal ; Clinically welll with normal renal function ; CaCl/CrCl =
<0.01
Neonatal severe PHPT
Multiple endocrine neoplasia
Hyperparathyroidism Jaw Tumor syndrome
Familial isolated PHPT
Malignancies
Local osteolytic hypercalcemia – with skeletal involvement
Humoral hypercalcemia – no skeletal involvement
69

Hypercalcemia
Pheochromocytoma
Acute adrenal insufficiency
Acromegaly
Thyroidism
Idiopathic Hypercalcemia of Infancy
Loss of Function mutations in CYP24A1 (25-Hydroxylvitamin D 24-hydroxylase)
Vitamin overdose (D and A)
Granulomatous Disease
Sarcoidosis, Tuberculosis, Berylliosis, Coccidioidomycosis
Renal Failure
CKD, AKI
70

Hypercalcemia
Milk-Alkali syndrome
End-stage liver disease
Multiple myeloma
Manganese intoxication
artificial nutrition (TPN)
Drugs
Calcium and Vitamin D and its analogs
PTH therapy
Estrogen or selective estrogen receptor modulators
Lithium
Growth hormone
Aminophylline / Theophylline
Chlorothiazide diuretics
71

Total Calcium Determination

Photometric Determination
o-cresolphthalein complexone method
Formation of red calcium chromophore complex measured at 570-580 nm
pH should be alkaline (pH 12.0)
Achieved by using organic bases like diethylamine, 2-amino-2methyl—propanol,
or 2-ethylaminoethanol
8-hydroxyquinolone – reduces interference caused by Mg2+
Urea may be added to reduce turbidity of lipemic samples and enhance complex
formation
Sodium acetate – increases linearity
Arsenazo III method
Formation of purple complex measured at acidic pH (6.0)
Buffer : imidazole
Measure at 650 nm
more specific to calcium than magnesium
72

Total Calcium Determination

Atomic Absorption Spectrometry – reference method
Direct / Indirect ISE
Specimen must be acidified to convert protein-bound and complexed calcium
Specimen Requirements
Do not use EDTA, Citrate, and oxalate
Interference from hemolysis when using photometric method
may be corrected by blanking with ethylene glycol-O-O’-bis(2-aminoethyl)-
N,N,N’,N’-tetraacetic acid (EGTA)
73

Ionized Calcium Determination

Direct / Indriect ISE : liquid membrane (ETH1001 or ETH129 carrier)
Specimen Requirements
Closed collection system
Sensitive to pH changes
Decreases by 5% for every 0.1 increase in pH
Heparinized whole blood – preferred ; analyzed in blood gas analyzers
Analyze within 15-30 mins of sampling
Lithium-zinc heparin – reduces effect of heparin on Ca2+
Do not place additional liquid heparin in heparinized syringes
Do not use EDTA, citrate, and oxalate.
74

Phosphate
Predominant intracellular anion
Exists in organic and inorganic forms
Only inorganic (H2PO4- and HPO42-) forms are measured
physiological pH ratio 1:4
Ratio 1:1 in acidic pH ; 1:9 in alkaline pH
Affected by circadian rhythm
Peak : evening after dinner
Nadir : early morning
FGF23 – mid to long-term modulator of phosphate homeostasis
Increases fractional excretion of phosphates in kidneys
Decreases production of calcitriol by inhibiting 25-hydroxyvitamin D 1-alpha-hydroxylase)
Secreted by bone cells
Responsiveness declines with kidney failure
Reference Interval
0.78 – 1.42 mmol/L
75

Hypophosphatemia
Serum inorganic phosphate < 0.78mmol/L
Relatively common in hospitalized patients
Not necessarily associated with intracellular depletion
Causes
Intracellular shift – common
Lowered Renal Phosphate Threshold
Decreased Net Intestinal Phosphate Absorption
Intracellular Loss
Drugs
76

Hypophosphatemia
Intracellular Shift
Refeeding syndrome – anabolic state when refeeding malnourished
Glucose / Fructose, Insulin – major cause
Diabetic ketoacidosis
Respiratory alkalosis – accelerates glycolysis
Alcoholism
Severe burns
Hungry bone syndrome
Renal Wasting
Hyperparathyroidism
Oncogenic osteomalacia
Renal tubular defects
Familial hypophosphatemia
Fanconi syndrome
X-linked hypophosphatemic rickets (XLH)
77

Hypophosphatemia
Inadequate Net Intestinal Absorption
Increased loss
Vomiting and diarrhea
Aluminum / magnesium containing antacids
Decreased absorption
Malabsorption syndrome
Vitamin D syndrome
Intracellular Loss
Acidosis
Hypophosphatemic Rickets – X-linked, Autosomal dominant, autosomal recessive
Dent disease
Vitamin D-resistant rickets
78

Drugs that cause Hypophosphatemia

Acetazolimide Estrogens
Anticonvulsants Ilfosamide
anti-EGF Iron polymaltose
HIV therapy Niacin
Azacitidine Paracetamol
Bisphosphonates Rapamycin
Catecholamines Salicylate
Cefottan Suramin
Diuretics Tyrosine-kinase inhibitors
79

Hyperphosphatemia
Serum inorganic phosphate > 1.42 mmol/L
Causes
Decreased Renal excretion
Increased Intake / Absorption
Increased Extracellular Load
Cell Lysis
Genetic
80

Hyperphosphatemia
Decreased RenalExcretion – most common cause
Decreased GFR : Renal Failure
Increased Tubular Reabsorption
Hypoparathyroidism
Pseudohypoparathyroidism
Acromegaly
Disodium etidronate
Increased extracellular Load
Transcellular shift
Acidosis
Untreated DKA
Malignant Hyperpyrexia
Increased Intake
Oral or IV
Laxatives or enemas
Vitamin D and its analogs
81

Hyperphosphatemia
Cell Lysis
Rhabdomyolysis
Intravascular hemolysis
Cytotoxic therapy
Tumor lysis syndrome
Leukemia
Lymphoma
Genetic
Tumoral calcinosis
Blomstrand disease
Epidermal naevus syndrome
osteoglophonic dysplasia
82

Phosphate Determination
Spectrophotometric
Fiske Subbarow Method
Most commonly used
Reagent : Ammonium molybdate
Formation of colorless phosphomolybdate complex measured at 340 nm
Carried at acidic pH
Fiske Subbarow with Reduction
Complex is reduced to molybdenum blue using
Pitcol (aminonaphthol sulfonic acid) - most common
Semidine (N-phenyl-p-phenyldoamine HCl)
Stannous chloride – provides greater color intensity
Measured at 600 – 700 nm

Do not use EDTA, oxalate or citrate!

83

Magnesium
2nd most prevalent intracellular cation
Distribution
55% - skeleton
45% - other tissues
<1% - blood
55% - free
30% - protein-bound
15% - complexed
Competitive inhibitor of Ca in the presynaptic nerve terminals
Vasodilator
Reference Intervals
Total serum Mg2+ : 0.66 – 1.07 mmol/L (1.7-2.4 mg/dL)
Free serum Mg2+ : instrument dependent
84

Hypomagnesemia
Common among hospitalized patients, mostly those in the ICUs
May cause neuromuscular hyperexcitability with tetany and seizures.
Gastrointestinal Disorders
Prolonged nasogastric suction
Malabsorption syndrome
Extensive bowel resection
Diarrhea
Fistulas
Acute hemorrhagic pancreatitis
Celiac disease
Primary hypomagnesemia (neonatal)
Renal loss
Osmotic diuresis
Glucose
Mannitol
Urea
Chronic parenteral therapy
85

Hypomagnesemia
Hypercalcemia
Phosphate depletion
Drugs – mainly alcohol and diuretics
Aminoglycosides
Proton pump inhibitors
Cardiac glycosides
Cisplastin
Cyclosporine
Amphotericin B
Pentamidin
Metabolic acidosis
Genetics
Barrter syndrome
Gittelman syndrome
86

Hypermagnesemia
Rarely encountered
Mainly caused by excessive intake
other causes
Chronic Renal Failure
Rhabdomyolysis associated with acute renal failure
Familial Benign hypocalciuric hypercalcemia
Lithium ingestion
87

Magnesium Determination
Colorimetric
Calmagite
formation of red-violet complex in alkaline pH measured at 530-550 nm
EGTA is used to chelate Ca
Formazan Dye
Formation of colored complex at alkaline pH measured at 630 nm using dry-slide
reflectance photometry
BAPTA is used to chelate calcium
Methylthymol Blue
Formation of blue complex measured at 600 nm
Magon or Xylidyl blue
Red complex at alkaline pH measured at 600 nm
AAS – reference method
Dye-Lake Method
Titan Yellow Dye
88
REFERENCES
Bishop, M. L., Fody, E. P., Schoeff, L. E. (2018). Clinical chemistry: Principles,
techniques, and correlations.

Cheng, S., Schindler, E., & Scott, M. (2018) Disorders of Water, Electrolytes, and Acid-
Base Metabolism. In Rifai, N., Horvath, A. R., & Wittwer, C. (Eds), Tietz
Textbook of Clinical Chemistry and Molecular Diagnostics (6th ed., pp.1324-
1333). Elsevier.

Fraser, W. (2018) Bone and Mineral Metabolism. In Rifai, N., Horvath, A. R., & Wittwer, C.
(Eds), Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (6th ed.,
pp.1422-1448). Elsevier.

Schindler, E., Brown, S., & Scott, M. (2018) Electrolytes and Blood Gases. In Rifai, N.,
Horvath, A. R., & Wittwer, C. (Eds), Tietz Textbook of Clinical Chemistry and
Molecular Diagnostics (6th ed., pp.604-615). Elsevier.