Kidney Function and Non-Protein Nitrogen Compounds PDF

CCHM311: CLINICAL CHEMISTRY 1 TOPIC: KIDNEY FUNCTION AND NON-PROTEIN NITROGEN COMPOUNDS 1ST SEMESTER | S.Y 2024-2025 LECTURER: Prof. Kimberly Ann M. Pulga, RMT, MPH TOPIC...

CCHM311: CLINICAL CHEMISTRY 1 TOPIC: KIDNEY FUNCTION AND NON-PROTEIN NITROGEN COMPOUNDS 1ST SEMESTER | S.Y 2024-2025 LECTURER: Prof. Kimberly Ann M. Pulga, RMT, MPH TOPIC KIDNEYS AND ITS GENERAL FUNCTION SUBTOPIC Retroperitoneal Excretory organ SUB SUBTOPIC Kidneys, ureter, urinary bladder, urethra 1. Elimination of metabolic waste products through the KIDNEY FUNCTIONS formation of urine - Kidneys are the major excretory organ of the body; Kidneys – 2 bean-shaped organs that are found in responsible for removal of waste and toxic vertebrates metabolites of cellular metabolic surplus (excess Located on the left and right in the retroperitoneal products) – eliminated by urine formation space otherwise accumulation of waste In adult humans: 11 cm length 2. Regulation of the plasma and water volume (most The basic structure involved in filtration and prominent) both ways and surplus reabsorption of materials in the body - They eliminate what is not essential [with the help - Received blood in the pair renal arteries; blood exits of ADH] into the paired renal veins 3. Regulation of ionic equilibrium Each kidney is attached to the ureter - Presence of electrolytes [sodium, calcium, Ureter – tube that carries excreted urine to the bladder magnesium, potassium, etc.] Nephron 4. Maintenance of acid-base balance - The functional unit of the kidney - Kidneys, together with the lungs, are the major - Responsible for urine formation organs that acts to achieve the optimal pH of the - Each kidney contain roughly 1.25 million functional body [which is around 7.4] units of the nephron o Majority of the metabolic processes will - Nephron is the basic structure involve in the actually operate once the body is in optimal filtration, secretion, and also the reabsorption of pH materials in the blood Glomerulus – region that supplies in the Bowman’s capsule, where the initial filtration happens Proximal Convoluted Tubule and Distal Convoluted Tubule – secretion and absorption Collecting duct – drainage from urine to the ureter Kidneys are responsible for the elimination of nitrogenous waste products - Removes end products of metabolism and other waste products of foreign substances dissolved in the plasma - Make adjustments in the concentration of normal constituents in the plasma 1|Page J.M.J.R  Kidneys are considered to be a minor endocrine organ; CLEARANCE TESTS it synthesizes and secretes hormones like EPO INULIN CLEARANCE TEST (Erythropoietin) Inulin freely passes the glomeruli but is neither - EPO (Erythropoietin) – targets Bone marrow to secreted nor reabsorbed by the tubules regulate the red cell maturation Considered to be the most accurate measure of GFR - Renin - enzyme-like substance that activates the Considered as a reference method of clearance test, RAAS system proper electrolytes and water balance but is not routinely done because it requires IV that stabilizes the blood pressure transfusion and requires multiple urine samples There are various kinds such as prostaglandin that is Priming Dose: 25 mL of 10% inulin solution produced by the kidney with aldosterone like functions Continuous Dose: 500 mL of 1.5% inulin Normal value: - 127 mL/min – men (due to larger renal mass) - 118 mL/min – women Normal values can be different from one laboratory to another laboratory, but the changes are not that significantly high - There can be deviations, pero hindi gaano kalayo CREATININE CLEARANCE TEST Most commonly used Provides an estimated amount of plasma that flows through urine into the kidney per minute Glomerular Filtration Not affected by diet Tubular Function – encompasses reabsorption and Directly related to the muscle mass of patient tubular secretion - We can use 24-hour urine sample Renal Blood Flow – consists of the non-protein nitrogen Excellent measure of the renal function, but this is compounds or NPN compounds not reabsorbed A measure of completeness of 24-hour urine TEST MEASURING GLOMERULAR FILTRATION RATE Normal Values: Consists of Clearance tests, Cystatin C, Beta-Trace - Male: 85-125 mL/min - Female: 75-112mL/min proteins, Myoglobin, and Microalbuminuria UREA CLEARANCE TEST GFR – Best overall indicator of the level of the kidney One of the oldest methods; oldest method function Demonstrates the progression of renal disease or in Tests that measure the rate of glomerular filtration are response on the therapy generally called Clearances freely filtered by the glomeruli - Inulin, creatinine, urea, etc. Urea is freely filtered by the glomeruli but variably In GFR, we are measuring the clearance of normal reabsorbed in the tubules depending upon the molecules not bound to protein and freely filtered by transmit time (rate of urine flow along the course of the glomerulus tubules) of urea filtrate The removal of substance from plasma into the urine Since the urea is reabsorbed it does not give reliable over a fixed time GFR, but is used to demonstrate progression of renal expressed in ml/minute, representing the volume of disease or response to therapy plasma that would be totally cleared of the solute in In advanced renal failure, the urea clearance is a one minute [mL/minute] better predictor of GFR compared to creatinine Plasma concentration is inversely proportional to clearance 2|Page J.M.J.R  CYSTATIN C PHENOLSULFONPHTALEIN (PSP) DYE EXCRETION TEST Measures the excretion of a dye proportional to renal Produced at a constant rate by all nucleated cells tubular mass Not secreted but completely reabsorbed Reference Range: 1200 mL/min Serum cystatin C levels are indirect estimates of GFR - Not secreted, but completely reabsorbed Once present in the urine, it denotes tubular damage CONCENTRATION TESTS Increases more rapidly than creatinine in the early stage SPECIFIC GRAVITY of GFR involvement Simplest test for renal concentrating ability or activity Not affected by muscle mass, age, diet, and gender Measurement is affected by solute number and mass number Reference values: - Reflects the function of the collecting ducts and - 0.5 to 1 (adults) loop of Henle - 0.9 to 3.4 (>64y/o) - We assess the quantity of solutes in the urine [concentrated urine or not] BETA TRACE PROTEINS High specific gravity = more concentrated [chloride, urea, sodium, etc.] Functions as prostaglandin C synthase Reference value: 1.005 – 1.030 Easily filtered and reabsorbed Specimen: First morning urine Isolated primarily from CSF OSMOLALITY Elevated in: Only affected by the number of solute present; - Myeloproliferative and lymphoproliferative represents the total number of solutes per kilogram disorders, inflammation, and renal failure of solvent Serum osmolality is due to sodium and chloride MYOGLOBIN Urine osmolality is due to ammonia Associated with acute skeletal and cardiac muscle injury Useful for assessing water deficits or excess Also termed as cardiac marker – we can use this one to Normal ratio of serum osmolality to urine osmolality assess acute myocardial infarction [MI or heart attack] is 1:1 - Increases during heart attack Reference values: - 275-295 mOsm/kg (serum) Rhabdmyolysis -> acute renal failure - 300-900 mOsm/kg (urine) MICROALBUMINURIA METHODS FOR MEASURING OSMOLALITY Small amount of albumin in the urine Direct Methods - Should not be present in the urine - Freezing Point Osmometry [most common] - Vapor Pressure Indirect Methods - Osmolal gap – difference between the measured and calculated plasma osmolality; TESTS MEASURING TUBULAR FUNCTIONS - A sensitive indicator of alcohol and drug overdose Serum Osmolality = 1.86 x Na x Glucose/18+BUN/ EXCRETORY TESTS 2.8 PARA-AMINO HIPPURATE TEST [PAH] OR DIODRAST TEST Measures the renal plasma flow Requires clearance of a dye Reference Range: 600-700 mL/min (administered through iodine intravenously) 3|Page J.M.J.R  NON-PROTEIN NITROGEN [NPN] BLOOD UREA NITROGEN [BUN] Non-Protein Nitrogen Compounds can be measured In severe liver damage levels of urea decrease. because they could indicate renal blood flow It is used as a screening test for kidney disease – since it Chemical substances that have low molecular weights – is the first metabolite to increase/elevate in kidney contain nitrogen, but not part of the protein molecules disease - Constitute products of catabolism of the proteins: Urea is readily removed by dialysis. urea, uric acid, and creatinine [and even ammonia] Example: Kidneys remove excess NPN of the body, so we can use - patient has normal to increase GFR – 40% BUN will NPN to evaluate the kidney or renal functions be reabsorbed; 60% will be excreted Usually expressed in mg/dL Well-hydrated patient = excretes more BUN - Resulting to serum or plasma lower BUN Dehydrated patient = BUN level decreases - Not excreting more of it 90% Urea is excreted; 10% remain in the blood - The remains is lost through the GI Tract or skin [remains in the blood] Clinical applications: - Evaluate renal function - Assess hydration status - Determine nitrogen balance - Aid in the diagnosis of renal diseases - Verify adequacy of dialysis UREA Major excretory product of protein metabolism First metabolite to increase during kidney diseases since it is a major organ solid in urine Only expressed by nitrogen content of urea (BUN) - There is no urea test, instead it is BUN Major nitrogen containing metabolic product of protein catabolism - Whether the protein is derived from endogenous or exogenous source, if they are being metabolized, there is urea High BUN = correlated to kidney disorder BUN to Creatinine Ratio = 10:1 to 20:1 Reference Value: 8-23 mg/dL (2.9-8.2 mmol/L) Conversion factor for BUN to Urea: 2.14 [multiply the factor 2.14 to convert the BUN to Urea] Conversion factor (mg/dL to mmol/L) = x0.357 [multiply by 0.36 (rounded off)] 4|Page J.M.J.R  We make use of enzymes here [enzymatic method] - Urease Coupled Urease/Glutamate Dehydrogenase - Use whole blood samples (GLD) Method – UV enzymatic - We have different methods under enzymatic - Indicator dye methods - Conductimetric Urea concentration can be measure using plasma ISOTOPE DILUTION MASS SPECTROMETRY – sample [in plasma sample, one must not utilize sodium REFERENCE METHOD citrate and sodium chloride because they inhibit the urease enzyme; enzymes function to hasten the chemical reaction], serum sample, or even urine sample Kinetic measurements – multiple measurements MICRO-KJELDAHL NESSLER IDMS – proposed reference method for urea Oldest method In the laboratory, we will perform enzymatic N → NH4 + alk. K2Hgl4 → NH2Hgl3 [quantitative enzymatic-colorimetric] - Double iodide of K and Hg - Making use of an indicator dye and measure the - Dimercuric ammonium iodide absorbance using the spectrophotometer Kjeldahl process - Absorbance will be correlated to the concentration - Mixture of sulfuric acid and phosphoric acid of urea in the sample - In NPN, there will be specific time intervals [kinetic measurements; not once, not twice, but some are CONSIDERATION IN BUN DETERMINATION thrice] Whole blood should be deproteinized to eliminate BLOOD UREA NITROGEN DETERMINATION interferences of hemoglobin [DIRECT MEASUREMENT OR CHEMICAL METHOD] Ammonium– containing anticoagulants are DIACETYL MONOXIME METHOD [DAM] contraindicated in enzymatic methods Urea is made to react with diacetyl monoxime to Sodium citrate and sodium chloride inhibits the action produce a yellow diazine derivative (Fearon’s of urease reaction) Upon prolonged standing, ammonia concentration in Arsenic thiosemicarbazide is added to enhance color the sample rises 2-3 times the original value due to formation and to exclude protein interference enzymatic deamination of labile amide like glutamine - After this, we make use of the - Process the specimen right away [all lab test – spectrophotometer to measure the intensity of process immediately] the absorbance - Directly proportional to the urea CLINICAL SIGNIFICANCE AZOTEMIA BLOOD UREA NITROGEN DETERMINATION Azotemia “azo” – nitrogen containing biochemical [INDIRECT MEASUREMENT OR ENZYMATIC METHOD] abnormality that refers to an increase in BUN and creatinine levels which is largely related to decrease Primary Step – hydrolysis of Urea by urease; production glomerular filtration of ammonia and carbon dioxide - Accompanied by renal failure = termed as uremia Urea + Urease → NH3 + CO2 [high creatinine levels] [highly fatal condition if - We will measure ammonia and carbon dioxide for no immediate treatment or intervention is the BUN determination provided] Measurement of Ammonia is most often used in - Increase concentration of BUN enzymatic methods: - Pre-renal azotemia [happens before kidney; decrease on renal blood flow = less blood - Urease-Nessler Method delivery in the kidney = less filtered urine], - Urease Berthelot Method azotemia, and post-renal azotemia 5|Page J.M.J.R  Azotemia is classified into three categories: CREATININE DETERMINATION 1. Pre-renal causes – conditions in which circulation through the kidneys is less efficient than usual Specimen Consideration: - Hemorrhage (blood loss) - Fasting sample is not required - Cardiac decompression - Serum and urine creatinine have both been used - Increased protein catabolism - 24-hour sample of urine with Tungsten blue bloodstream and joints. Uses Sodium Cyanide as a color stabilizer - Definitive diagnosis-presence of birefrigent crystals in the synovial fluid. MODIFICATION OF CARAWAY - Chronic alcoholism increases uric acid in the blood Sodium carbonate is used for the determination of stream because alcohol inhibits its excretion. uric acid - Predominantly found in men than women UA + PTA -> Tungsten blue - Symptoms: Uses Sodium Carbonate as color stabilizer o Arthritis, painful or inflammation of joints, INDIRECT MEASUREMENT [ENZYMATIC METHOD] nephrolithiasis, and in tissues particularly BLAUNCH AND KOCK OR URICASE METHOD the big toe Differential or Absorption Spectrophotometry - Treatment: Uric acid is destroyed by the action of uricase to form carbon dioxide and allantoin. Since uric acid has a o No alcohol maximum absorption peak at 290-293nm, white o Proper diet and weight maintenance allantoin has no absorption at this point, the o Drug therapy if wanted decrease in the absorbance is positively related to o Allopurinol – drug used to treat gout the uric acid present in the sample. Leukemia and other malignant conditions due to Uric acid is converted to allantoin and carbon dioxide increased turnover of nucleoproteins and there is a peak absorbance [290nm-293nm], but Uric acid levels are elevated in decreased renal not applicable to allantoin functions either due to over production of uric acid or Lower absorbance = higher uric acid conc decrease rate of excretion. Uses special quartz cuvette in performing this - Can also occur in patients with cancer or malignant method conditions like chemotherapy and radiation therapy IDMS or Isotope Dilution Mass Spectrometry – that can cause increased cell destruction – high proposed reference method levels of purines Fatal poisoning with chloroform and methanol, excessive exposure to X-rays and radioactive radiators 8|Page J.M.J.R  due to excessive cell breakdown and nucleic acid catabolism Radiation and chemicals Genetic diseases like Lesch-Nyhan syndrome-deficiency in hypoxanthine-guanine phosphoribosyl transferase(HGPRT) - Responsible for the behavioral changes of an individual like pathological aggressiveness and self- mutilation QUESTIONS What is the major end product of protein and amino acid metabolism? - Blood Urea Nitrogen Which of the following clearance test is considered to be the reference method? - Inulin clearance test After the urease reaction, ammonia produced can be treated with _________. - Berthelot reagent In this method, serum is mixed with alkaline picrate and the rate of change in absorbance is measure between 2 points. - Kinetic Jaffe Method This is the reference method for measuring creatinine: - IDMS This refers to elevated plasma urea and other nitrogenous waste products accompanied by azotemia and electrolyte imbalance - Pre-renal azotemia [before the kidney] Disease primarily in males, accompanied by pain and inflammation of the joints and is diagnosed with birefringent crystal in synovial fluid - Gout Excretion test/s: - Either para-amino Hippurate test and diodrast test 9|Page J.M.J.R