This document describes a method for determining total fatty acids in serum through microtitration. The key steps are: 1) Extracting lipids from serum using chloroform-methanol, 2) Saponifying the extracted lipids using alcoholic potassium hydroxide to liberate fatty acids, 3) Extracting the liberated fatty acids using hexane, 4) Directly titrating the hexane phase containing fatty acids with sodium hydroxide using a color indicator. The method allows determination of total fatty acids, cholesterol, and phospholipids from 1mL of serum and achieves 97% recovery of pure fatty acids and triglycerides.
This document outlines a presentation on renal handling of urea, uric acid, and creatinine. It begins with objectives to explain renal clearance and discuss handling of the three substances. Renal clearance is introduced as the rate at which a solute is removed from the body by excretion or metabolism. Urea is 50% reabsorbed in the proximal tubule and its excretion varies with urine flow rate. Uric acid freely filters but is almost entirely reabsorbed and then secreted back in the proximal tubule. Creatinine is neither reabsorbed nor secreted and its clearance can estimate glomerular filtration rate.
The document discusses various methods of calculating drug clearance from the body. It describes physiologic clearance which is the rate of drug flow through an organ cleared per unit time. Renal clearance is the volume of plasma cleared of drug by the kidneys per unit time. Graphical and model independent methods allow direct calculation of clearance from plasma concentration-time curves without assumptions. Factors like drug properties, binding, blood flow, disease states can impact renal clearance. Total body clearance is the sum of individual organ clearances.
The document discusses how urine pH affects the tubular reabsorption and renal excretion of weak acids and bases. It states that acidification of urine promotes reabsorption of weak acids and retards reabsorption of weak bases, while alkalization of urine has the opposite effects. The extent of ionization of drugs depends on the drug's pKa value and urine pH. It also discusses how urine flow rate and drug clearance influence renal drug excretion. Clearance is defined as the volume of fluid cleared of drug per unit of time and can be used to determine the mechanism of renal drug excretion.
This document discusses clearance concepts in pharmacokinetics. It defines clearance as the proportionality constant relating the rate of drug elimination to its concentration in blood or plasma. Clearance depends on blood flow and the extraction ratio of the clearing organ. The two main types of clearance are hepatic clearance and renal clearance. Renal clearance can be estimated by measuring creatinine clearance. The document provides equations for calculating clearance, area under the curve, and creatinine clearance and adjusting drug doses based on renal function.
The document discusses clearance and renal excretion of drugs. It defines clearance as the volume of fluid cleared of the drug per unit time. Renal clearance is the volume of plasma cleared of the drug by the kidneys per unit time. The kidney is the major organ of excretion. Renal clearance can provide information about the mechanisms of renal excretion such as filtration, secretion, and reabsorption. The clearance concept is used to describe the elimination of drugs by various organs.
Renal clearance is a measurement of the volume of plasma from which a substance is completely removed by the kidney per unit time, usually per minute. It reflects the kidney's rate of drug elimination divided by plasma concentration. The total body clearance is equal to renal clearance plus hepatic and lung clearance. Renal clearance allows analysis of kidney activity and is defined as the volume of plasma from which a substance is completely removed by the kidney in a given time. Glomerular filtration rate can be measured by clearance techniques using marker substances that are filtered but not reabsorbed or secreted by the kidneys, such as inulin or creatinine.
The document provides information on respiratory and reproductive pharmacology. For respiratory pharmacology, it discusses animal models used in in-vitro and in-vivo tests, including histamine receptor binding and the effects of arachidonic acid. For reproductive pharmacology, it discusses animal models used to study mineralocorticoid activity through electrolyte excretion tests and progestational activity through progesterone receptor binding assays. The document provides details on the procedures, evaluations, and modifications of these pharmacology tests in respiratory and reproductive systems.
This document outlines a presentation on renal handling of urea, uric acid, and creatinine. It begins with objectives to explain renal clearance and discuss handling of the three substances. Renal clearance is introduced as the rate at which a solute is removed from the body by excretion or metabolism. Urea is 50% reabsorbed in the proximal tubule and its excretion varies with urine flow rate. Uric acid freely filters but is almost entirely reabsorbed and then secreted back in the proximal tubule. Creatinine is neither reabsorbed nor secreted and its clearance can estimate glomerular filtration rate.
The document discusses various methods of calculating drug clearance from the body. It describes physiologic clearance which is the rate of drug flow through an organ cleared per unit time. Renal clearance is the volume of plasma cleared of drug by the kidneys per unit time. Graphical and model independent methods allow direct calculation of clearance from plasma concentration-time curves without assumptions. Factors like drug properties, binding, blood flow, disease states can impact renal clearance. Total body clearance is the sum of individual organ clearances.
The document discusses how urine pH affects the tubular reabsorption and renal excretion of weak acids and bases. It states that acidification of urine promotes reabsorption of weak acids and retards reabsorption of weak bases, while alkalization of urine has the opposite effects. The extent of ionization of drugs depends on the drug's pKa value and urine pH. It also discusses how urine flow rate and drug clearance influence renal drug excretion. Clearance is defined as the volume of fluid cleared of drug per unit of time and can be used to determine the mechanism of renal drug excretion.
This document discusses clearance concepts in pharmacokinetics. It defines clearance as the proportionality constant relating the rate of drug elimination to its concentration in blood or plasma. Clearance depends on blood flow and the extraction ratio of the clearing organ. The two main types of clearance are hepatic clearance and renal clearance. Renal clearance can be estimated by measuring creatinine clearance. The document provides equations for calculating clearance, area under the curve, and creatinine clearance and adjusting drug doses based on renal function.
The document discusses clearance and renal excretion of drugs. It defines clearance as the volume of fluid cleared of the drug per unit time. Renal clearance is the volume of plasma cleared of the drug by the kidneys per unit time. The kidney is the major organ of excretion. Renal clearance can provide information about the mechanisms of renal excretion such as filtration, secretion, and reabsorption. The clearance concept is used to describe the elimination of drugs by various organs.
Renal clearance is a measurement of the volume of plasma from which a substance is completely removed by the kidney per unit time, usually per minute. It reflects the kidney's rate of drug elimination divided by plasma concentration. The total body clearance is equal to renal clearance plus hepatic and lung clearance. Renal clearance allows analysis of kidney activity and is defined as the volume of plasma from which a substance is completely removed by the kidney in a given time. Glomerular filtration rate can be measured by clearance techniques using marker substances that are filtered but not reabsorbed or secreted by the kidneys, such as inulin or creatinine.
The document provides information on respiratory and reproductive pharmacology. For respiratory pharmacology, it discusses animal models used in in-vitro and in-vivo tests, including histamine receptor binding and the effects of arachidonic acid. For reproductive pharmacology, it discusses animal models used to study mineralocorticoid activity through electrolyte excretion tests and progestational activity through progesterone receptor binding assays. The document provides details on the procedures, evaluations, and modifications of these pharmacology tests in respiratory and reproductive systems.
The aim of the use of meloxicam in patients with mild or moMUSHTAQ AHMED
This document summarizes several studies on the pharmacokinetics of the drug meloxicam. It finds that meloxicam has a half-life of around 20 hours, making it suitable for once-daily dosing. It is metabolized into inactive metabolites that are excreted in urine and feces. The pharmacokinetic profile in rats most closely resembles that in humans. Studies also show that dosage adjustment is not needed for meloxicam in patients with mild to moderate renal impairment or in the elderly.
Pancreatic function tests can diagnose diseases of the exocrine pancreas or exocrine pancreatic insufficiency. Tests for diseases include serum amylase, serum lipase, and sweat chloride. Amylase and lipase levels rise in acute pancreatitis as the enzymes leak from damaged pancreatic cells. Sweat chloride testing diagnoses cystic fibrosis by measuring abnormally high chloride levels caused by a genetic mutation. Tests for exocrine pancreatic insufficiency include direct measurement of enzymes in pancreatic juice and indirect tests of fat and elastase levels in stool samples.
This document discusses renal clearance, which is the volume of plasma cleared of drug per unit of time by the kidneys. Renal clearance is determined graphically by plotting the rate of drug excretion in urine against the plasma drug concentration to calculate the slope, which equals clearance. Factors that can affect renal clearance include physicochemical drug properties, plasma concentration, distribution/binding characteristics, urine pH, blood flow to the kidneys, and disease states. The concept of clearance is now applied to describe drug elimination by organs like the liver, lungs and bile.
B. Pharm. (Honours) Part-III Practical, Pharmacology II,MANIKImran Nur Manik
a) Estimation of blood glucose by enzymatic method.
b) Estimation of blood glucose by chemical method.
c) Estimation of aspirin after oral administration by UV spectrophotometric method.
d) Estimation of aspirin after oral administration by calorimetric method.
e) Estimation of plasma protein by enzymatic method.
f) Estimation of plasma protein by burette method.
g) Estimation of blood uric acid level by enzymatic method.
h) Estimation of Paracetamol after oral administration by UV/Visible spectrophotometric method.
i) Handling of experimental animals: mice and rat.
j) Different routes of administration of drugs in experimental animals.
k) Assay of serum SGOT and SGPT activities in mice.
l) Assay of serum alkaline phosphatase activity
m) Isolation and determination of cholesterol content of biological samples.
B. Pharm. (Honours) Part-IV Practical, Pharmacology-III, MANIKImran Nur Manik
3. Pharmacology-III: (Marks-30)
Estimation of glucose in blood in normal condition and after administration of insulin; biological assay of digitalis, histamine and insulin; microbiological assay of antibiotics and vitamins; spectrophotometric estimation of blood pigments; toxicity test of the drugs like, phenobarbitone, nikethamide, some antineoplastic drugs, pilocarpine, etc.
Pocket Guide: Pharmacokinetics Made Easy is the latest update of the popular Pharmacokinetics Made Easy. It is suitable for a wide audience including medical practitioners, health professionals, and students. The individual chapters were initially published as a series of articles in Australian Prescriber to assist practitioners in drug dosing and therapy. The physiological approach herein adopted addresses clinical issues in drug therapy and makes them directly applicable to practice situations.
This document discusses drug elimination, which occurs through metabolism and excretion. Excretion involves the irreversible transfer of drugs and metabolites from the internal to external environment through two main routes: renal and non-renal excretion. Renal excretion occurs primarily through the kidneys via glomerular filtration, active tubular secretion, and active/passive tubular reabsorption. These processes work to either increase or decrease drug concentration in the kidney tubules and thereby facilitate or prevent drug elimination from the body. Factors like a drug's physicochemical properties, distribution, binding characteristics, urine pH, blood flow to the kidneys, and disease states can impact renal clearance and excretion.
This document summarizes renal function in the ICU, including calculation of glomerular filtration rate (GFR) and creatinine clearance, fractional excretion of sodium, hepatorenal syndrome, rhabdomyolysis, and contrast-induced acute renal failure. It also presents several case studies involving evaluation of renal function and diagnosis of conditions such as acute tubular necrosis, SIADH, and normal renal function based on creatinine clearance calculations and urinary indices.
Clinical chemistry uses chemical processes to measure levels of chemical components in the blood. It is very useful for the early diagnostic of disease and for monitoring organ function. The most common specimens used in clinical chemistry are blood and urine. Table 1 shows the common blood tests and measurable items using UV/Vis spectrophotometers.In this application note, the cholesterol level in human serum was determined by the enzymatic method using the LAMBDA™ 465 UV/Vis Spectrophotometer and UV Lab™ software.
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
This document provides information on estimating serum cholesterol levels, including:
- What cholesterol is and how it is produced in the body
- Symptoms of high cholesterol and how it can lead to atherosclerosis and heart disease
- Methods for estimating total cholesterol using the Zak's method or enzymatic method
- Classification and functions of lipoproteins such as LDL, HDL, VLDL
- Desirable cholesterol levels according to NCEP-ATP III guidelines
- Protocol for estimating total cholesterol and HDL cholesterol levels
Estimation of glucose in blood in normal condition and after administration of insulin; biological assay of digitalis, histamine and insulin; microbiological assay of antibiotics and vitamins; spectrophotometric estimation of blood pigments; toxicity test of the drugs like, phenobarbitone, nikethamide, some antineoplastic drugs, pilocarpine, etc.
This document summarizes drug excretion, which is the removal of drugs from the body through various routes. The major routes of excretion are the kidneys (renal excretion), bile/liver (biliary secretion), lungs (pulmonary excretion), and feces. Renal excretion occurs through glomerular filtration, tubular reabsorption, and tubular secretion in the kidneys. Biliary secretion involves the active transport of drugs from liver cells into bile. Pulmonary excretion eliminates gaseous and volatile substances through exhaled breath. Factors like a drug's properties, age, disease states, and urine pH can impact the rate of excretion.
This document discusses renal clearance, which is the volume of plasma cleared of drug per unit time through the kidneys. Renal clearance is affected by physicochemical drug properties, plasma concentration, distribution, binding, urine pH, blood flow to kidneys, and disease states. Physiological processes involved in renal clearance include glomerular filtration, tubular secretion, and tubular reabsorption. The clearance ratio compares a drug's clearance to inulin clearance to determine the excretion mechanism. Renal clearance can be determined graphically or using model-independent methods involving the amount of drug excreted in urine and area under the plasma concentration-time curve.
This document discusses cholesterol, including its importance, metabolism, extraction, and reduction in beef suet using lecithin. It provides details on cholesterol's properties, sources, and transport in the blood. Methods for estimating total cholesterol are described, such as spectrophotometry and gas chromatography. Cholesterol content is listed for various fish, shellfish, and mollusks. The document also examines bleaching and deodorizing beef suet, purifying soybean lecithin, and using lecithin to reduce cholesterol in beef suet by forming a lecithin-cholesterol complex. Greater cholesterol removal was achieved with higher lecithin-to-suet and stirring ratios.
This document discusses linear and nonlinear pharmacokinetics. Linear pharmacokinetics follow first-order kinetics and nonlinear pharmacokinetics follow Michaelis-Menten kinetics. Nonlinearity can occur due to saturation of drug absorption, distribution, metabolism or excretion processes. The Michaelis-Menten equation can describe nonlinear kinetics and data plots of drug concentration versus time can indicate nonlinear behavior.
This document discusses glomerular filtration and its regulation. It begins with an introduction to the kidney and nephron. There are three main processes involved in urine formation: glomerular filtration, tubular reabsorption, and tubular secretion. Glomerular filtration is governed by the filtration coefficient and Starling forces, including hydrostatic and oncotic pressures. The glomerular filtration rate can be measured and is regulated by various intrinsic and extrinsic mechanisms, including the myogenic response, tubuloglomerular feedback, neural inputs, and hormones like angiotensin II and atrial natriuretic peptide. Conditions like exercise, pregnancy, posture, and disease states can influence glomerular filtration rate.
In Vitro Metabolism of Clonidine in Human Hepatic Microsomes and Cytochrome P...Claessens
1. The document identifies the primary metabolite of clonidine as hydroxyclonidine and develops an LC-MS method to quantify both compounds using a deuterated internal standard.
2. It characterizes the kinetic parameters of clonidine hydroxylation in human liver microsomes and screens 17 cytochrome P450 isoforms, finding five that hydroxylate clonidine with varying potency.
3. Selective inhibition experiments show that CYP2D6, CYP1A2, and CYP3A4/5 contribute most to clonidine hydroxylation in human liver microsomes.
1. The document describes a laboratory experiment to estimate blood glucose levels using the glucose oxidase/Trinder's method. Glucose in blood samples is oxidized by glucose oxidase to produce hydrogen peroxide, which is measured colorimetrically.
2. Three blood samples were tested and their glucose concentrations calculated. Sample 3 had the highest concentration at 11.34 mmol/L, indicating hyperglycemia. Sample 2 was within the normal range, while Sample 1 was hypoglycemic at 2.75 mmol/L.
3. The results confirm the visual observation that Sample 3 appeared most colored, correctly identifying hyperglycemia in that sample based on the quantitative analysis. The method allows accurate glucose measurement to
Ameliorating Effect of Frankincense on Red Blood Cells of Alloxan Induced-Dia...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
This document discusses cough and various drugs used to treat cough. It explains that cough is a protective reflex to clear the respiratory tract. Chronic cough can cause fatigue and should be treated. The cough reflex involves central and peripheral nervous systems and bronchial smooth muscle. Expectorants work by thinning mucus to make it easier to cough up, while cough suppressants work centrally or peripherally to reduce cough reflex. The document discusses various expectorants like bromhexine, acetylcysteine, carbocysteine, and guaifenesin, and cough suppressants like codeine, dextromethorphan, and benzodiazethine. It provides details on their mechanisms and recommended doses.
The triglyceride level test measures the amount of triglycerides in the blood. Triglycerides are a type of fat that provides energy and high levels can increase the risk of heart disease. The test is ordered to determine heart disease risk, estimate LDL cholesterol, check for pancreas inflammation, and assess risk of atherosclerosis. Doctors recommend getting tested every 5 years as part of a regular checkup, more often if being treated for high triglycerides or if diabetic. Elevated levels may be due to lifestyle factors like smoking or diet, or medical conditions like diabetes, and can be controlled through diet and exercise changes.
The aim of the use of meloxicam in patients with mild or moMUSHTAQ AHMED
This document summarizes several studies on the pharmacokinetics of the drug meloxicam. It finds that meloxicam has a half-life of around 20 hours, making it suitable for once-daily dosing. It is metabolized into inactive metabolites that are excreted in urine and feces. The pharmacokinetic profile in rats most closely resembles that in humans. Studies also show that dosage adjustment is not needed for meloxicam in patients with mild to moderate renal impairment or in the elderly.
Pancreatic function tests can diagnose diseases of the exocrine pancreas or exocrine pancreatic insufficiency. Tests for diseases include serum amylase, serum lipase, and sweat chloride. Amylase and lipase levels rise in acute pancreatitis as the enzymes leak from damaged pancreatic cells. Sweat chloride testing diagnoses cystic fibrosis by measuring abnormally high chloride levels caused by a genetic mutation. Tests for exocrine pancreatic insufficiency include direct measurement of enzymes in pancreatic juice and indirect tests of fat and elastase levels in stool samples.
This document discusses renal clearance, which is the volume of plasma cleared of drug per unit of time by the kidneys. Renal clearance is determined graphically by plotting the rate of drug excretion in urine against the plasma drug concentration to calculate the slope, which equals clearance. Factors that can affect renal clearance include physicochemical drug properties, plasma concentration, distribution/binding characteristics, urine pH, blood flow to the kidneys, and disease states. The concept of clearance is now applied to describe drug elimination by organs like the liver, lungs and bile.
B. Pharm. (Honours) Part-III Practical, Pharmacology II,MANIKImran Nur Manik
a) Estimation of blood glucose by enzymatic method.
b) Estimation of blood glucose by chemical method.
c) Estimation of aspirin after oral administration by UV spectrophotometric method.
d) Estimation of aspirin after oral administration by calorimetric method.
e) Estimation of plasma protein by enzymatic method.
f) Estimation of plasma protein by burette method.
g) Estimation of blood uric acid level by enzymatic method.
h) Estimation of Paracetamol after oral administration by UV/Visible spectrophotometric method.
i) Handling of experimental animals: mice and rat.
j) Different routes of administration of drugs in experimental animals.
k) Assay of serum SGOT and SGPT activities in mice.
l) Assay of serum alkaline phosphatase activity
m) Isolation and determination of cholesterol content of biological samples.
B. Pharm. (Honours) Part-IV Practical, Pharmacology-III, MANIKImran Nur Manik
3. Pharmacology-III: (Marks-30)
Estimation of glucose in blood in normal condition and after administration of insulin; biological assay of digitalis, histamine and insulin; microbiological assay of antibiotics and vitamins; spectrophotometric estimation of blood pigments; toxicity test of the drugs like, phenobarbitone, nikethamide, some antineoplastic drugs, pilocarpine, etc.
Pocket Guide: Pharmacokinetics Made Easy is the latest update of the popular Pharmacokinetics Made Easy. It is suitable for a wide audience including medical practitioners, health professionals, and students. The individual chapters were initially published as a series of articles in Australian Prescriber to assist practitioners in drug dosing and therapy. The physiological approach herein adopted addresses clinical issues in drug therapy and makes them directly applicable to practice situations.
This document discusses drug elimination, which occurs through metabolism and excretion. Excretion involves the irreversible transfer of drugs and metabolites from the internal to external environment through two main routes: renal and non-renal excretion. Renal excretion occurs primarily through the kidneys via glomerular filtration, active tubular secretion, and active/passive tubular reabsorption. These processes work to either increase or decrease drug concentration in the kidney tubules and thereby facilitate or prevent drug elimination from the body. Factors like a drug's physicochemical properties, distribution, binding characteristics, urine pH, blood flow to the kidneys, and disease states can impact renal clearance and excretion.
This document summarizes renal function in the ICU, including calculation of glomerular filtration rate (GFR) and creatinine clearance, fractional excretion of sodium, hepatorenal syndrome, rhabdomyolysis, and contrast-induced acute renal failure. It also presents several case studies involving evaluation of renal function and diagnosis of conditions such as acute tubular necrosis, SIADH, and normal renal function based on creatinine clearance calculations and urinary indices.
Clinical chemistry uses chemical processes to measure levels of chemical components in the blood. It is very useful for the early diagnostic of disease and for monitoring organ function. The most common specimens used in clinical chemistry are blood and urine. Table 1 shows the common blood tests and measurable items using UV/Vis spectrophotometers.In this application note, the cholesterol level in human serum was determined by the enzymatic method using the LAMBDA™ 465 UV/Vis Spectrophotometer and UV Lab™ software.
Haemolysis effect of Mefenamic Acid 250 mg Capsule in Bio analysis by liquid ...IOSR Journals
A rapid, simple and specific method for estimation of Mefenamic acid in human plasma was validated using Indomethacin as internal standard. The analyte and internal standard were extracted from plasma using simple solid phase extraction. The compound were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mM Ammonium Acetate in Water and acetonitrile (20:80, v/v) and detected by tandem mass spectrometry in negative ion mode. The ion transition recorded in multiple reaction monitoring mode were m/z 240.1 196.0 for Mefenamic acid and m/z 356.1312.0 for internal standard. Linearity in plasma was observed over the concentration range 35.000 – 7000.000 ng/mL for Mefenamic acid. The cv of the assay was 4.89 % to 5.98 % and accuracy was 99.36 to 102.20 % Intra and Interday respectively at LLOQ level. The validated method was applied to bioequivalence study of 250 mg Mefenamic acid in 28 healthy human volunteers. Total 50 samples from individual volunteers identified as Haemolyzed which were analyze initial and repeat again to cross check the method reproducibity for Haeamolysis effect and compared which found acceptable range
This document provides information on estimating serum cholesterol levels, including:
- What cholesterol is and how it is produced in the body
- Symptoms of high cholesterol and how it can lead to atherosclerosis and heart disease
- Methods for estimating total cholesterol using the Zak's method or enzymatic method
- Classification and functions of lipoproteins such as LDL, HDL, VLDL
- Desirable cholesterol levels according to NCEP-ATP III guidelines
- Protocol for estimating total cholesterol and HDL cholesterol levels
Estimation of glucose in blood in normal condition and after administration of insulin; biological assay of digitalis, histamine and insulin; microbiological assay of antibiotics and vitamins; spectrophotometric estimation of blood pigments; toxicity test of the drugs like, phenobarbitone, nikethamide, some antineoplastic drugs, pilocarpine, etc.
This document summarizes drug excretion, which is the removal of drugs from the body through various routes. The major routes of excretion are the kidneys (renal excretion), bile/liver (biliary secretion), lungs (pulmonary excretion), and feces. Renal excretion occurs through glomerular filtration, tubular reabsorption, and tubular secretion in the kidneys. Biliary secretion involves the active transport of drugs from liver cells into bile. Pulmonary excretion eliminates gaseous and volatile substances through exhaled breath. Factors like a drug's properties, age, disease states, and urine pH can impact the rate of excretion.
This document discusses renal clearance, which is the volume of plasma cleared of drug per unit time through the kidneys. Renal clearance is affected by physicochemical drug properties, plasma concentration, distribution, binding, urine pH, blood flow to kidneys, and disease states. Physiological processes involved in renal clearance include glomerular filtration, tubular secretion, and tubular reabsorption. The clearance ratio compares a drug's clearance to inulin clearance to determine the excretion mechanism. Renal clearance can be determined graphically or using model-independent methods involving the amount of drug excreted in urine and area under the plasma concentration-time curve.
This document discusses cholesterol, including its importance, metabolism, extraction, and reduction in beef suet using lecithin. It provides details on cholesterol's properties, sources, and transport in the blood. Methods for estimating total cholesterol are described, such as spectrophotometry and gas chromatography. Cholesterol content is listed for various fish, shellfish, and mollusks. The document also examines bleaching and deodorizing beef suet, purifying soybean lecithin, and using lecithin to reduce cholesterol in beef suet by forming a lecithin-cholesterol complex. Greater cholesterol removal was achieved with higher lecithin-to-suet and stirring ratios.
This document discusses linear and nonlinear pharmacokinetics. Linear pharmacokinetics follow first-order kinetics and nonlinear pharmacokinetics follow Michaelis-Menten kinetics. Nonlinearity can occur due to saturation of drug absorption, distribution, metabolism or excretion processes. The Michaelis-Menten equation can describe nonlinear kinetics and data plots of drug concentration versus time can indicate nonlinear behavior.
This document discusses glomerular filtration and its regulation. It begins with an introduction to the kidney and nephron. There are three main processes involved in urine formation: glomerular filtration, tubular reabsorption, and tubular secretion. Glomerular filtration is governed by the filtration coefficient and Starling forces, including hydrostatic and oncotic pressures. The glomerular filtration rate can be measured and is regulated by various intrinsic and extrinsic mechanisms, including the myogenic response, tubuloglomerular feedback, neural inputs, and hormones like angiotensin II and atrial natriuretic peptide. Conditions like exercise, pregnancy, posture, and disease states can influence glomerular filtration rate.
In Vitro Metabolism of Clonidine in Human Hepatic Microsomes and Cytochrome P...Claessens
1. The document identifies the primary metabolite of clonidine as hydroxyclonidine and develops an LC-MS method to quantify both compounds using a deuterated internal standard.
2. It characterizes the kinetic parameters of clonidine hydroxylation in human liver microsomes and screens 17 cytochrome P450 isoforms, finding five that hydroxylate clonidine with varying potency.
3. Selective inhibition experiments show that CYP2D6, CYP1A2, and CYP3A4/5 contribute most to clonidine hydroxylation in human liver microsomes.
1. The document describes a laboratory experiment to estimate blood glucose levels using the glucose oxidase/Trinder's method. Glucose in blood samples is oxidized by glucose oxidase to produce hydrogen peroxide, which is measured colorimetrically.
2. Three blood samples were tested and their glucose concentrations calculated. Sample 3 had the highest concentration at 11.34 mmol/L, indicating hyperglycemia. Sample 2 was within the normal range, while Sample 1 was hypoglycemic at 2.75 mmol/L.
3. The results confirm the visual observation that Sample 3 appeared most colored, correctly identifying hyperglycemia in that sample based on the quantitative analysis. The method allows accurate glucose measurement to
Ameliorating Effect of Frankincense on Red Blood Cells of Alloxan Induced-Dia...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
This document discusses cough and various drugs used to treat cough. It explains that cough is a protective reflex to clear the respiratory tract. Chronic cough can cause fatigue and should be treated. The cough reflex involves central and peripheral nervous systems and bronchial smooth muscle. Expectorants work by thinning mucus to make it easier to cough up, while cough suppressants work centrally or peripherally to reduce cough reflex. The document discusses various expectorants like bromhexine, acetylcysteine, carbocysteine, and guaifenesin, and cough suppressants like codeine, dextromethorphan, and benzodiazethine. It provides details on their mechanisms and recommended doses.
The triglyceride level test measures the amount of triglycerides in the blood. Triglycerides are a type of fat that provides energy and high levels can increase the risk of heart disease. The test is ordered to determine heart disease risk, estimate LDL cholesterol, check for pancreas inflammation, and assess risk of atherosclerosis. Doctors recommend getting tested every 5 years as part of a regular checkup, more often if being treated for high triglycerides or if diabetic. Elevated levels may be due to lifestyle factors like smoking or diet, or medical conditions like diabetes, and can be controlled through diet and exercise changes.
Lecture Xv Arteriosclerosis And Atherosclerosis 11Amelia Monteiro
The document discusses problems with oxygen transport and arterial diseases like arteriosclerosis and atherosclerosis. It covers lipids like cholesterol and complexes like lipoproteins that can contribute to blocked arteries. Risk factors for atherosclerosis are described like age, genetics, smoking, and high serum lipids. Types of angina and diagnostic tests for coronary artery disease are outlined. Management strategies are also summarized, including diet, exercise, medication, and procedures like angioplasty and CABG.
Cardiac output is the volume of blood pumped by the heart per minute. It is calculated as heart rate multiplied by stroke volume. Cardiac output can vary depending on the body's activity level and is regulated by factors that influence heart rate and stroke volume. The Frank-Starling law of the heart states that increased venous return leads to increased stretch of the heart muscle and increased force of contraction, resulting in higher stroke volume and cardiac output.
Cardiac output is defined as the volume of blood pumped by the heart each minute and is regulated intrinsically by factors affecting preload and afterload as well as extrinsically by the autonomic nervous system and hormones. Venous return is a primary extrinsic regulator of cardiac output, increasing stretch of cardiac muscles and stimulating an increase in heart rate. A combination of preload, contractility, afterload and heart rate determine cardiac output under normal resting conditions and during physical activity.
Arteriosclerosis is the hardening and narrowing of arteries due to plaque buildup. The three main types are arteriolosclerosis (small arteries), Monckeberg medial sclerosis (calcification of muscular arteries), and atherosclerosis (most common). Atherosclerosis features atheromas that protrude into the vessel lumen. Risk factors like age, gender, genetics, hyperlipidemia, hypertension, smoking, and diabetes accelerate atherosclerosis. Inflammation and infection also contribute to plaque formation and rupture, which can cause acute issues like heart attack or stroke.
This document discusses methodology for lipid profile analysis, including cholesterol, lipoproteins, triglycerides, and apolipoproteins. It covers pre-analytical considerations for specimen collection and storage, as well as analytical methods such as enzymatic, chemical, and electrophoretic techniques. Cholesterol is measured using enzymatic cholesterol oxidase or chemical Liebermann-Burchardt reactions. Lipoproteins are separated by polyanion precipitation, electrophoresis, or ultracentrifugation. Triglycerides are analyzed chemically or enzymatically using glycerol kinase. Quality control is also important in lipid analysis.
ACTH, also known as corticotropin, is a polypeptide hormone secreted by the pituitary gland that stimulates the production of cortisol by the adrenal glands. Two common bioassays are described for measuring ACTH activity. The first involves measuring ascorbic acid depletion in the adrenal glands of hypophysectomized rats after ACTH administration. The second measures corticosterone levels in the blood of dexamethasone-blocked rats at increasing time points after ACTH injection. Both assays involve administration of multiple doses of a standard ACTH preparation and test preparation to rats, followed by quantitative chemical analysis to determine potency ratios and confidence limits for the test preparation relative to the standard
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Cholesterol is one of the most studied molecules in biology. It plays essential roles in animal cell membranes and is a precursor for bile acids, steroid hormones, and vitamin D. Cholesterol is synthesized endogenously through a complex multi-step process and is also obtained through diet. High levels of cholesterol are linked to atherosclerosis and heart disease, while adequate levels are important for various biological functions. Tight regulation of cholesterol homeostasis is necessary for health.
The document summarizes key information about renal function tests. It discusses how the kidneys filter waste and regulate fluid balance and electrolytes. Common renal function tests measure blood urea and creatinine levels, which are elevated when kidney function declines. Creatinine clearance and glomerular filtration rate are calculated to estimate kidney function. Jaffe and kinetic methods are used to measure creatinine levels colorimetrically. Normal ranges are provided for various tests.
This document summarizes key functions and tests related to kidney and renal function. It discusses that the kidneys filter waste from the blood, regulate fluid balance and electrolytes, and produce hormones. Renal function can be assessed by blood urea and creatinine levels, which increase with reduced kidney function. Creatinine clearance and glomerular filtration rate are tests that evaluate kidney filtration rate and are useful for detecting and monitoring kidney disease.
The kidneys play a vital role in the excretion of waste products and toxins such as urea, creatinine and uric acid, regulation of extracellular fluid volume, serum osmolality and electrolyte concentrations, as well as the production of hormones like erythropoietin and 1,25 dihydroxy vitamin D and renin.
Specimen collection requirements are dependent on the procedure or test requested. Generally, for serum creatinine and blood urea nitrogen (BUN) levels, no additional patient preparation is required, and a random blood sample suffices. However, the effect of recent high protein ingestion may increase serum creatinine and urea levels to a significant extent. Also, hydration status can have a considerable impact on BUN measurement.
For timed urine collections such as the 24-hour urine creatinine clearance, it is essential that urine be collected accurately over the required period as under or over collection will affect final results. Hence, a 5 to 8-hour timed collection is preferable to a 24-hour collection.
There are several clinical laboratory tests that are useful in investigating and evaluating kidney function. Clinically, the most practical tests to assess renal function is to get an estimate of the glomerular filtration rate (GFR) and to check for proteinuria (albuminuria).
Tests of renal function can be used to assess overall renal function by direct measurement or estimation of the glomerular filtration rate. Estimation of the GFR is utilized to determine the presence of renal impairment.
This document summarizes key functions and tests related to kidney and renal function. It discusses that the kidneys filter waste from the blood, regulate fluid balance and electrolytes, and produce hormones. Renal function can be assessed by blood urea and creatinine levels, which increase with reduced kidney function. Creatinine clearance and glomerular filtration rate are tests that evaluate kidney filtration rate and are useful for detecting and monitoring kidney disease.
Metabolism of stability studies and sub cellular fractionsManjunatha D C
This document provides information on metabolic stability studies using cellular and sub-cellular fractions. It discusses hepatocyte and sub-cellular fraction isolation procedures for rats and mice. Metabolic stability is defined as the percentage of parent compound lost over time when exposed to metabolically active systems like hepatocytes, liver microsomes, or liver S9 fractions. Isolated hepatocytes and sub-cellular fractions like cytosol and microsomes are used to study hepatic drug metabolism, uptake, and clearance mediated by drug-metabolizing enzymes and transporters. Detailed methods are provided for isolating hepatocytes and hepatic macrophage from rat and mouse liver, as well as preparing sub-cellular fractions from rat and mouse liver.
The researchers aimed to purify cellular retinol binding protein (CRBP) from bovine liver. Through a process involving homogenization, centrifugation, cation exchange chromatography, gel filtration, and concentration, they obtained a final product. However, characterization through SDS-PAGE and absorption spectroscopy identified the protein as catalase rather than CRBP. Despite initial absorption at 350nm for CRBP, the maximal absorption and thermal/pH profiles matched those of catalase. The purification resulted in the isolation of catalase rather than the intended CRBP.
This document describes laboratory procedures for measuring various biomarkers related to cholesterol and cardiac health. It discusses the principles, reagents, and procedures for measuring creatine kinase, cholesterol, triglycerides, HDL cholesterol, and LDL cholesterol. Creatine kinase measurement involves detecting isoforms that indicate cardiac injury. Cholesterol measurement uses extraction and a colorimetric reaction. Triglycerides are measured via enzymatic reactions producing a colored product. Precipitation separates lipoproteins before measuring HDL and LDL cholesterol levels.
Investigating Novel Methods to Reduce Cholesterol Levels (Research Report)Tony Ng
In this study, we used everyday food items such as lactic acid bacteria (found in yogurt), sunflower seeds and kidney beans, to either absorb cholesterol in the gut or inhibit cholesterol synthesis in the body. Findings can potentially provide doctors and health experts with an alternative to statins, cholesterol-lowering drugs that may lead to side effects. As future work, we can explore the chemical compound present in the natural extracts that help inhibit cholesterol synthesis, and if possible, carry out trials in vivo to better determine the effects of these extracts.
Lipids are digested and absorbed in a multi-step process. Dietary lipids are broken down by lingual and gastric lipases in the mouth and stomach. In the small intestine, bile salts emulsify lipids and pancreatic lipase further breaks them down. Fatty acids and monoglycerides are absorbed via micelle formation and enterocytes, where they are reassembled into triglycerides and packaged into chylomicrons for transport through lymphatic vessels and blood. Chylomicrons deliver lipids to tissues via interaction with cell receptors and lipoprotein lipase.
This document summarizes lipid digestion and metabolism. It discusses how lipids are not digested in the mouth or stomach but are broken down by enzymes in the small intestine. Bile salts produced by the liver emulsify lipids and activate pancreatic lipase. Lipids are hydrolyzed into fatty acids and monoglycerides which are absorbed via micelles into intestinal cells. There they are re-esterified and packaged into chylomicrons which enter the lymphatic system and blood. Chylomicrons deliver lipids to tissues while liver produces VLDL to export endogenous lipids. VLDL and chylomicrons exchange triglycerides for cholesterol to become LDL and HDL which circulate lipids through the body
The document describes a human anti-hemophilic vaccine produced from human plasma. It discusses the collection of plasma from healthy donors, the production process which involves precipitation and purification using column chromatography, and the assay used to determine potency by comparing clotting times. The production involves testing donor blood to ensure safety, separating plasma from blood within 12 hours, and using an ion exchange column to capture factor VIII. The purified fraction is tested for activity and virus inactivation is performed before lyophilization.
Biochemistry of Kidney-5 and 6.pdthfjdfhrtfSriRam071
The kidney regulates homeostasis through filtration and reabsorption. Each kidney contains approximately one million nephrons, the functional units of the kidney. During filtration, 120-125 mL of plasma is filtered into the glomerular filtrate per minute. Over 99% of the filtrate is reabsorbed by the renal tubules. Tests of kidney function include clearance tests to assess glomerular filtration rate (GFR) such as creatinine clearance, which estimates the volume of plasma cleared of creatinine per minute based on creatinine levels in a 24-hour urine collection and plasma sample.
a) Estimation of blood glucose by enzymatic method.
b) Estimation of blood glucose by chemical method.
c) Estimation of aspirin after oral administration by UV spectrophotometric method.
d) Estimation of aspirin after oral administration by calorimetric method.
e) Estimation of plasma protein by enzymatic method.
f) Estimation of plasma protein by burette method.
g) Estimation of blood uric acid level by enzymatic method.
h) Estimation of Paracetamol after oral administration by UV/Visible spectrophotometric method.
i) Handling of experimental animals: mice and rat.
j) Different routes of administration of drugs in experimental animals.
k) Assay of serum SGOT and SGPT activities in mice.
l) Assay of serum alkaline phosphatase activity
m) Isolation and determination of cholesterol content of biological samples.
This document discusses lipid metabolism and fatty acid synthesis and oxidation. It begins by explaining that triacylglycerols (TGs), consisting of fatty acids esterified to glycerol, are the main form in which lipids are stored in the body as an energy reserve. The pathways of fatty acid synthesis and oxidation, which occur in the cytoplasm and mitochondria respectively, are then outlined. Key processes like elongation, desaturation, and the carnitine shuttle for transporting long-chain fatty acids into mitochondria are also summarized. Alternative pathways for unsaturated fatty acids and the production of ketone bodies during fasting are briefly mentioned.
This document summarizes different intravenous (IV) fluid options used in intensive care, including crystalloids, colloids, and specific fluid products. Crystalloids like saline readily diffuse out of blood vessels, while colloids like albumin, hetastarch, and pentastarch remain in circulation longer due to their larger size. Albumin is the main protein in blood plasma and expands volume the least of colloids. Hetastarch is a synthetic starch that expands volume more than albumin but can cause coagulopathy in large doses. Pentastarch is a newer low-molecular-weight hetastarch derivative that may cause fewer side effects.
1. Volume 1
Number 1
The microtitration of total fattv acids ot serum,
with notes on the estimation of triglycerides*
MARGARETJ. ALRRINK~
Department of Internal Medicine, Yale University
School of Medicine, New Haven 11, Connecticut
[Received for publication June 22, 19591
S U M M A R Y
A method is described for the determination of total fatty acids of serum by microtitration.
The method is designed to permit also the determination of cholesterol and lipid phosphorus,
and of triglycerides by difference. One ml. of serum is sufficient for duplicate determinations
of total fatty acids, lipid phosphorus, and cholesterol. The essential steps include extraction
of serum lipids, saponification,extraction and microtitration of the liberated fatty acids. A POI+
sible modification which may be useful in the estimation of triglycerides is described. Re-
coveries of pure fatty acids and triglycerides were 97 per cent complete. Short chain fatty
acids and intermediates of carbohydrate metabolism were not detected by this method.
F a t t y acids normally represent about 50 per
cent of the total serum lipids. The fatty acids occur
chiefly in phospholipids, esters of cholesterol, and in
triglycerides, but small moieties also are present as
di- and monoglycerides and as free fatty acids bound
to albumin. No single lipid constituent accurately rep-
resents the status of the other lipids. For clinical as
well as research purposes a lipid analysis is not com-
plete unless the three major classes of lipids are meas-
ured. The concentration of total fatty acids provides
an index of total lipids in terms of their common de-
nominator, and when determined with cholesterol and
phospholipids, permits by calculation estimation of
triglycerides. The latter are of significance in coro-
nary artery disease, diabetes, and other metabolic dis-
orders as well as in the physiological transport of fat
(1 to 4), Since chemical reactions are best expressed
as taking place between equivalent weights rather
than absolute weights of compounds, the concentra-
tion of total fatty acids is most usefully exp&d in
terms of chemical equivalents rather than absolute
weight. A need thus exists for a practical yet accurate
method for determining total fatty acids as equiva-
lents, preferably a method sharing as many steps as
possible with the chemical procedures necessary for
determination of cholesterol and phospholipids.
*Aided in part by United States Public Health Service
tThis work was done during the tenure of an Established
Grant H-3498Cof the National Heart Inatitute.
Investigatorship of the American Heart Association.
Titrimetric determination of total fatty acids after
hydrolysis of serum lipids permits direct estimation of
total fatty acids as equivalents. With modern micro
apparatus 4 ml. of serum seems disadvantageous and
excessive for duplicate determinations of cholesterol,
phospholipids, and fatty acids measured by the Man
and Gildea modification of the Stoddard and Drury
method ( 5 ) .
The present method provides a technique for the
microtitrimetic determination of total fatty acids of
small amounts of serum. The method is of sufficient
simplicity to permit its use as a routine procedure,
yet accurate enough for use as a research tool. It was
designed to permit also the determination of choles-
terol by the method of Abell et al. (6) and of phos-
pholipids by the Sperry method (7). One ml. of serum
is sufficient for duplicate determinations of these three
substances, although less than two-tenths of a ml. of
serum is actually used for duplicate titrations of total
fatty acids alone.
The lipids are initially extracted by the method of
Folch et al. (8) with certain modifications. There fol-
lows a saponification procedure similar to, but more
intensive than, that recommended by Abell et al. for
total cholesterol (6). Following acidification of the
saponified lipids, the liberated fatty acids as well as
cholesterol are extracted by shaking with petroleum
ether or hexane. The final step is the direct micro-
titration of the fatty acids in a hexane-alcohol two-
phase system similar to that which has been described
53
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2. 54 ALBRINK J. Lipid Researah
October, 1059
by Dole (9) for the titration of “nonesterified fatty
acids.”
M E T H O D S
Reagents.
Chloroform (reagent grade) ,redistilled twice weekly.
Methanol (reagent grade),redistilled twice weekly.
Hexane (reagent grade). The Fisher product is used
without further purification.
Absolute ethyl alcohol (reagent grade). U.S. Indus-
trial Chemical Company absolute alcohol is used
without further purification.
Chloroform-methanol (exactly 2:1 v/v) .
Alcoholic potassium hydroxide: 6 cc. of 33 per cent
potassium hydroxide are added to 94 CC. of absolute
alcohol daily (6).
1per cent phenolphthalein in 95 per cent alcohol.
1.8 N hydrochloric acid.
Nile blue indicator: 0.02 per cent aqueous Nile blue
A solution is washed four or five times with hexane,
then diluted 1:10 with absolute alcohol (10).
Standard 0.02 N sodium hydroxide.
Extraction. All determinations are carried out in
duplicate. The procedure for a single determination
is described. One half ml. of serum (or plasma) is
pipetted into a 50 ml. round bottom glass-stoppered
centrifuge tube.l Exactly 10 ml. of 2:l chloroform-
methanol are added to the serum with constant and
vigorous swirling during the addition. The centrifuge
tube is then stoppered, sealed with a drop of water,
and shaken gently with a back-and-forth motion for
1minute. The chloroform-methanol-serum mixture is
allowed to stand at room temperature with occasional
gentle agitation for 1hour. Ten ml. of distilled water
are then added gently down the side of the tube. The
tube is stoppered without further shaking and allowed
to stand in the refrigerator overnight, or for several
days if desired. A reagent blank, included in each
run, is prepared by the addition of 10.5 ml. distilled
water to 10 ml. chloroform-methanol and treated the
same way as the sera throughout. After 15 hours the
mixture has separated into a lower chloroform phase
and an upper aqueous phase which contains impuri-
ties of the original mixture soluble in methanol and
water. The precipitated proteins are largely concen-
trated as a disk between the upper and lower phases.
Most of the lower chloroform phase is removed
through a long metal cannula2 or long hypodermic
needle attached to a 10 ml. hypodermic syringe. The
1 Corning centrifuge tubes #8424 are excellent.
2 Becton-Dickinson stainleM steel laboratory cannula, #B-D
1250NR.
cannula is passed carefully through the aqueous phase
and the protein precipitate by sliding it down the wall
to the bottom of the centrifuge tube. About 5.5 ml.
of the 6.67 ml. of clear chloroform extract can be re-
moved and transferred to a test tube from which ali-
quots are then taken. At this stage it is convenient
to remove 1 ml. for lipid phosphorus determination
(7) if desired. From the remaining chloroform extract,
4 ml. are measured into a clean 50 ml. glass-stoppered
centrifuge tube for determination of cholesterol and
total fatty acids.
Saponification. The chloroform is readily evapo-
rated from the unstoppered centrifuge tubes by plac-
ing the tubes in a large vacuum desiccator equipped
with a sand bath which has been preheated to 50°C.
The desiccator is evacuated by a water aspirator
pump; 45 minutes is usually sufficient to bring the ex-
tracts to dryness. Within a few minutes of the comple-
tion of the evaporation (the lipids must not be left dry)
5 ml. of alcoholic potassium hydroxide and 1 drop of
phenolphthalein are added to the dried lipid extract in
the centrifuge tubes. The tubes are incubated in a water
bath at 80°C for 1hour. Approximately 5 ml. of water
are then added to each tube. After 10 more minutes in
the water bath, 1ml. of 1.8normal aqueous hydrochloric
acid is added to the warm hydrolysate and the mix-
ture swirled. This is a little more than enough hydro-
chloric acid to neutralize the potassium hydroxide and
should decolorize the phenolphthalein and precipitate
the liberated fatty acids as a fine cloud.
When the mixture has cooled to room temperature,
exactly 10 ml. of hexane are added to each tube. The
tube is stoppered, sealed with a drop of water, shaken
vigorously for 1minute, and centrifuged for 5 minutes
at about 2000 rpm. The clear upper hexane phase then
contains virtually all of the liberated fatty acids and
the cholesterol now as free cholesterol.
If cholesterol is determined, an appropriate aliquot
(5 ml. is satisfactory) may now be pipetted from the
upper hexane phase for cholesterol determination by
the method of Abell et al. ( 6 ) .Then 3 ml. are pipet-
ted from the remaining hexane phase into a 15 ml.
centrifuge tube for determination of total fatty acids.
Rubber bulb Propipettesm have been found useful in
removing the aliquots from the upper phase without
disturbing the lower phase.
Microtitration of Total Fatty Acids. Now 3 ml. of
Nile blue indicator are added to the 3 ml. of hexane
extract in a 15 cc. centrifuge tube. This two-phase sys-
tem, an upper hexane phase containing the fatty acids
and a lower alcohol phase containing the indicator, is
titrated directly by adding 0.02 normal NaOH from a
microburette. The 1 ml. Gilmont ultramicroburette
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3. VolumeNumber11 MICROTITRATION OF SERUM TOTAL FATTY ACIDS 55
has been found satisfactory. For about 2 minutes be-
fore and also during the titration a stream of nitrogen,
which has been through a solution of 300 ml. of indi-
cator with 40ml. of 33 per cent NaOH to remove car-
bon dioxide, is bubbled through the hexane and indi-
cator through a fine glass tube leading to the bottom
of the centrifuge tube. This serves to mix the two
phases and to exclude carbon dioxide from the air.
The end point is indicated by a change in color from
blue to pink.
Calculations. The concentration of total fatty acids
in meq. per 1. is calculated according to the following
formula:
Total fatty acids in meq./l.
= (ml. of 0.02 N NaOH - blank)
1000
ml. serum extracted
x 0.02X
total chloroform total hexane
chloroform aliquot hexane aliquot
Example: 0.5 ml. of serum was extracted with 10 ml.
of 2:l chloroform methanol. After removal of the
methanol, 4 ml. of the 6.67 ml. of chloroform were re-
moved for total fatty acid and cholesterol determina-
tion, and the solvent evaporated. After saponification,
and addition of 10 ml. of hexane, 3 ml. of the hexane
were measured for titration of total fatty acids. The
unknown required a titer of 0.0755 ml. of 0.02 N so-
dium hydroxide; the blank gave a titer of 0.0080 (the
blank is almost entirely due to the indicator and is
constant from day to day). Using the above formula,
it was determined that the concentration of total fatty
acids in the serum was 15.0 meq. per 1.
XX
1000 6.67 10
0.5 4 3
(0.0755 - 0.0080) X -x 0.02 x -x -
= 15.0meq./l.
Calculation of Triglycerides. The triglyceride con-
centration expressed as milliequivalents of triglycer-
ide fatty acids per liter is calculated by subtracting
the fatty acids of cholesterol esters and phospholipids
from the total fatty acids.
The cholesterol is assumed to be 72 per cent esteri-
fied (11, 12).This ratio varies between the narrow
limits of 68 to 76 per cent in the sera of normal per-
sons (11). The assumed value of 72 per cent would
therefore be within 6 per cent of the actual value. Al-
though small increases in the ratio of esterified choles-
terol to total cholesterol are common in diabetic acido-
sis and essential hyperlipemia, the triglycerides are also
increased so that any error incurred by the use of the
above formula would be small compared to the total
triglycerides (4). In the presence of liver disease or ob-
structive jaundice, however, the ratio of esterified cho-
lesterol to total cholesterol may be greatly reduced
(13) and the freecholesterol should be measured rather
than assumed. Unless the serum is obviously icteric,
the above formula can be used with impunity.
The phospholipid fatty acids are estimated by as-
suming that 20 per cent of the phospholipids have
one fatty acid molecule per atom of phosphorus
(sphingomyelin) and that the remaining 80 per cent
have two fatty acid molecules for every phosphorus
atom (lecithin and cephalins) (11). The assumption
regarding sphingomyelin has been borne out by Phil-
lips’ recent report (14) that sphingomyelin constitutes
18 per cent of normal serum phospholipids.
The fatty acids not accounted for as cholesterol es-
ters or phospholipids are assumed to be derived from
triglycerides, recognizing that a small fraction of these
are present as free fatty acids or as mono- and di-
glycerides (9, 10, 15). The following formula is used,
the derivation of which has been discussed previously
(11):
Triglyceride fatty acids in meq./l. = total fatty acids
in meq./l. - (cholesterol fatty acids
+phospholipid fatty acids),
where cholesterol fatty acids in meq./l. =
10 X 0.72 X total cholesterol in mg../100 ml.
386
and phospholipid fatty acids in meq./l. =
10 X l(0.80 X 2) +0.21lipid phosphorus in mg./100 ml.
31
R E S U L T S
Reproducibility. The difference between duplicate
titrations was usually less than 0.0020ml. of 0.02N
sodium hydroxide. In the entire range of total fatty
acids tested, the mean difference between duplicate
determinations was 0.40meq. per 1. f0.37 meq. per 1.
(or 1.98 f 1.4per cent). As practice is gained with the
microburette, this error is rarely exceeded.
Recoveries. The completeness of recovery of fatty
acids was tested by carrying out the entire procedure
on solutions containing known concentrations of pure
fatty acids or triglyceride.s The material to be tested
was dissolved in hexane in known concentrations. An
appropriate volume of the hexane solution was meas-
3 Obtained from The Hormel Foundation, Austin, Minn.
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4. 56 ALBRINK J. Lipid Research
Oclober, 1858
ured into a 50 ml. centrifuge tube and the solvent re-
moved in vacuo.To the dried lipid was added 0.5 ml.
of water if the material was to be recovered alone, or
0.5 ml. of serum if it was to be recovered from serum.
The results were calculated as concentration in water
(or serum) and are thus directly comparable with the
results of analyses of sera.
The recoveries of palmitic acid and triolein, alonc
or added to serum, were respectively 97.9 f2.6 per
cent, and 98 f3.7 per cent. Lauric acid was also 97
per cent recovered. The concentrations tested ranged
from 8to 100meq. per l., a range encompassing all nor-
mal and most abnormal sera. In concentrations below 8
meq. per l., recoveries were less complete. Since aliquots
of the same size were used for all determinations, the
method has applicability over a wide range of concen-
tration without need to adjust the size of aliquots.
Recovery of fatty acids with shorter chain length than
lauric was not investigated because to date these have
not been found in appreciable amounts in serum (16).
The possibility that short-chain organic acids might
interfere with the method was ruled out by analyzing
aqueous solutions of pyruvic, citric, aceto-acetic, suc-
cinic, and lactic acids in concentrations between 100
and 200 mg. per 100ml., higher than are likely to occur
in serum. The values obtained were indistinguishable
from the reagent blank.
As a further test of the method, fatty acid determi-
nations were made on 60 sera which had been deter-
mined in the laboratory of Dr. Evelyn B. Man by the
method of Man and Gildea (5). These sera were ob-
tained from both norma1 persons and patients with a
variety of disorders associated with abnormal lipids,
including several cases of hypercholesterolemia and
essential hyperlipemia. The fatty acid concentration3
in these sera ranged from 6 to 100 meq. per 1. and the
titers of 0.02 N sodium hydroxide ranged from 0.027
to 0.45 ml. The mean difference between methods was
0.80 meq. per l., f 1.0. When the difference between
pairs was expressed as percentage of the total fatty
acids, the mean difference was 3.6 f 3.1 per cent. The
positive deviations almost exactly equaled the negative
deviations, so that the average algebraic difference
was only 0.005 meq. per 1.
If the lipid phosphorus is not determined, or is meas-
ured directly on serum, the total fatty acids can be
measured by adding 5 ml. of the alcoholic potassium
hydroxide directly to 0.5 ml. of serum and proceeding
with the saponification and titration as described. The
serum must be swirled during addition of the alcoholic
potassium hydroxide, but further shaking only causes
clumping of the protein and should be avoided.
The cholesterol method used here was that of Abell
et al. (6), except that the serum lipids were extracted
and dried prior to saponification, acidified, and ex-
tracted with hexane rather than petroleum ether after
saponification. The extraction procedure was thought
to be desirable prior to saponification in order to pro-
vide a common starting point for the determinations
of total fatty acids, cholesterol, and phospholipids.
Recovery of cholesterol added to serum prior to ex-
traction was complete. In 15 sera analyzed by the
method of Abell et al., both with and without prior
extraction, the cholesterol concentrations averaged 7
mg. per 100 ml. or 2.6 per cent higher in the extracted
sera, although care was taken to treat the standards
in exactly the same manner as the unknowns. This
discrepancy has not been explained but is less than
the error of the method (3 per cent).
A comparison between the cholesterol concentra-
tions determined by the method cited here and by the
gravimetric determination (17) from an alcohol-ether
extract as conducted in the laboratory of Dr. Evelyn
B. Man revealed that the gravimetric method gave
somewhat lower values. The absolute amount of the
discrepancy increased as the concentration of choles-
terol increased; the relative discrepancy remained
constant at about 7 to 9 per cent. This difference is
small compared to the day-by-day fluctuations of
serum cholesterol in a given individual. The lower
gravimetric determinations may reflect mechanical er-
rors of this technique, which may be greater now
than before, since imported sintered glass funnels for-
merly used may have retained cholesterol digitonide
more completely than the domestic funnels now in use.
Nonphospholipid Fatty Acids. The indirect estima-
tion of triglycerides can be reduced in cumulative
errors by eliminating the phospholipids, and thus any
possible errors in assuming the fraction of total fatty
acids contributed by the phospholipids. Advantage is
taken of the absorption of phospholipids by silicic
acid (18,19) in the following adaptation of the method
of Eder.4
The serum lipids are extracted by adding to 0.5 ml.
serum 10 ml. of 2 :1 chlorofonn-methanol, followed
by 10 ml. of water, as described in the section on ex-
traction. After standing overnight, about 5.5 ml. of
the clear chloroform layer are transferred to a fresh
50 ml. centrifuge tube, with the aid of a syringe and
long cannula, care being taken not to transfer any
water with the chloroform. Approximately 0.5 g. of
silicic acid (1/4 level teaspoon household measure is
4 H. A. Eder. Unpublished method.
5 Silicic acid (Mallinckrodt) 100 mesh, analytical reagent,
“activated”by heating in an oven at 105°Cfor 24 hours.
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5. Volume 1
Number I MICROTITRATION OF SERUM TOTAL FATTY ACIDS 57
convenient and measures about 0.44 g. silicic acid)
is added. to the chloroform extract through a funnel,
with gentle agitation during the addition. The glass
stopper is promptly inserted and after standing 15
minutes, with frequent brief, gentle agitation, the tube
is centrifuged for 10 minutes. Exactly 4 ml. of the
clear chloroform supernatant is pipetted into a fresh
50 ml. centrifuge tube. The subsequent evaporation,
saponification, acidification, extraction with hexane,
and measurement of aliquots for cholesterol and total
fatty acid determination, are identical with the pro-
cedures described in the section on methods, except
that 50 minutes is sufficient for saponification. The
fatty acid titer represents the nonphospholipid fatty
acids and is composed of triglyceride and cholesterol
ester fatty acids. The triglyceride fatty acids are cal-
culated by subtracting the cholesterol fatty acids (see
above) from the nonphospholipid fatty acids.
The completeness of recovery of triglycerides by
this method was assessed by analyzing solutions of
pure triolein, varying in concentration from 8 to 60
meq. per l., and treated exactly as serum, and 96 to
100per cent of the triolein was recovered. Ten per cent
of free fatty acids added (as palmitic) was lost, but
the contribution of free fatty acids to total fatty acids
is so small that this loss would not have a detectable
influence on the concentration of nonphospholipid
fatty acids. Treatment with silicic acid did not influ-
ence the reagent blank.
Twenty sera of 20 different persons covering a wide
range of concentrations of cholesterol, lipid phos-
phorus, and triglycerides were analyzed both with and
without the addition of silicic acid to the chloroform
extract. In none of the 20 was there detectable phos-
phorus in the extract treated with silicic acid. The
cholesterol concentration was not changed by treat-
ment with silicic acid. The triglycerides calculated by
the two methods are shown in Table 1.There is good
agreement between the two methods over the entire
range examined. However, there is a distinct tendency
for the triglycerides to be higher in the silicic acid
method. This suggests either that there is more
sphingomyelin present than assumed in the formula
for calculating triglycerides, or that phospholipids are
incompletely extracted or saponified during the de-
termination of total fatty acids. In either event, the
silicic acid method is probably the method of choice
for future studies.
D I S C U S S I O N
The extraction procedure described by Folch et al.
(8)and adapted to serum by Sperry et al. (20) has been
TABLE 1. LIPIDVALUESOF TWENTYNORMALAND ABNORMAL
SERA: TRIGLYCERIDESCALCULATEDIN THE CONVENTIONAL
WAYBY DIFFERENCEAND AZTERREMOVALOF PHOSPHO-
LIPIDS WITH SILICICACID
TriglycerideFatty Acids
Serum
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Cholesterol
mg./lOO ml.
348
194
410
369
282
286
362
232
310
217
396
210
273
274
147
171
307
231
342
395
Lipid P
ng./lOOml.
11.6
8.4
18.5
11.5
12.0
12.1
12.1
8.8
20.8
10.7
16.1
9.2
11.6
10.5
6.0
8.0
11.5
11.6
15.0
15.1
Zonventional
meqll.
4.1
4.5
53.3
5.6
12.3
6.8
8.7
2.8
52.1
4.6
18.0
6.3
8.6
7.0
2.9
2.8
5.1
5.6
19.1
2.6
After Silicic
Acid
meq./1.
4.7
3.7
53.2
6.1
14.5
8.2
10.2
3.4
54.0
5.9
20.2
5.3
8.5
9.5
3.3
3.1
5.7
7.2
18.9
3.3
simplified in several respects. First, the separate ad-
dition of the chloroform and methanol and the heating
of the extract (20) were not found necessary to ensure
adequate extraction. It was, however, found essential
to swirl constantly during, and for a brief period after,
the addition of chloroform-methanol to the serum in
order to ensure fine dispersion of the protein precipi-
tate in the extraction mixture.
A second modification of the procedure of Folch
et al. is the elimination of the filtration of the protein
precipitate. The cold chloroform is of such great den-
sity that the proteins float to the top of the chloro-
form phase as the methanol diffuses into the upper
aqueous phase.
A third modification avoids the removal of the
aqueous phase and subsequent washings, as well as
the reconstitution of the remaining chloroform with
methanol and the necessity of adding solvent to a
definite volume before taking aliquots. The modifica-
tion is based on the assumption that all the chloroform
originally added in the chloroform-methanol mixture
is quantitatively restored by the removal of the
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6. 58 ALBRINK J. Lipid Research
October, 1959
methanol into the aqueous phase or that traces of
chloroform lost to the upper phase are balanced in
volume by traces of water and methanol remaining
in the lower phase. The measurement of volumes in
a long 50 ml. glass-stoppered cylinder graduated in
0.02 ml. divisions, showed that a volume equal to the
20 ml. of chloroform theoretically present in 30 ml.
of 2:1 chloroform-methanol mixture was restored by
mixing with 30 ml. of water. Under these circum-
stances the evaporation of chloroform is evidently neg-
ligible, for the level of the chloroform water interface
remained unchanged after standing at room tempera-
ture for 3 months. Changes in laboratory temperature
between 20" and 30°C caused negligible volume
changes.
Because of the possibility that serum free fatty
acids, a small constituent of total fatty acids, might
be incompletely recovered (21) unless the aqueous
layer were acidified (22), dilute sulfuric acid and 0.5
M sodium acid phosphate buffer (pH = 4.02) were
tried in place of water. In neither instance was the
recovery of total fatty acids changed in the least.
The saponification is essentially that recommended
by Abell et al. (6) for hydrolysis of cholesterol esters.
Increasing the temperature from 38" to 80°C increased
the yield of total fatty acids, presumably by improving
the hydrolysis of the phospholipids. Increasing the
strength of the alcoholic potassium hydroxide from ap-
proximately 2 per cent to 15per cent or prolonging the
time of saponification to 5 hours produced no further
increase in total fatty acids. Acidification of the sapon-
ification mixture after incubation, necessary for extrac-
tion of the liberated fatty acids by hexane, made no dif-
ference in the cholesterol determination and did not
contribute to the fatty acid reagent blank. The quanti-
tative recovery of the hexane from the hexane-alcohol-
water mixture was ascertained by the measurement
of appropriate volumes in a graduated cylinder.
The ingenious two-phase titration system used by
Dole for titration of free fatty acids (9) was satis-
factory, and because the concentration of total fatty
acids is so much greater than the concentration of
free fatty acids, much smaller amounts of serum
were required. The Nile blue indicator recommended
by Gordon (10) gave a good end point. The lag in
color change, presumably caused by the lag in trans-
fer of fatty acids between phases, was minimized by
the use of hexane instead of heptane and by increas-
ing the volume of indicator to equal the volume of
hexane solution. The titer of the blank was almost
entirely contributed by the indicator, and constituted
as much as 20 per cent of the titer of the unknown.
Great care was taken, therefore, in the daily titration
of the blank and in the accurate addition of the indi-
cator. Lower blanks and better checks were obtained
when carbon-dioxide-free nitrogen was allowed to
bubble through the test tube for a few minutes prior
to titrati0n.O
Standardization of the alkali was carried out by
weekly titration against dilutions of 0.1 N hydro-
chloric acid, and the correction factor thus obtained
incorporated into each calculation. The burette was
filled with sodium hydroxide from a small glass-stop-
pered bottle filled freshly each day, and flushed out
with nitrogen before filling.
The present method has been found readily adapt-
able to the determination of lipids in lipoprotein frac-
tions as well as in whole serum or plasma. It conserves
time since little extra manipulation is required over
that necessary for determining lipid phosphorus and
cholesterol. When these determinations are also made,
the present method compares favorably in both time
and accuracy with existing methods, such as the hy-
droxylamine colorimetric method (22, 23). It might
complement the triglyceride method of Van Handel
and Zilversmit (24). It gives more chemically specific
information than the gravimetric method of total lipid
analysis (20), and avoids the assumptions necessary
in the colorimetric total lipid method of Bragdon (25).
Finally, it gives results closely comparable with those
obtained by the method of Man and Gildea ( 5 ) . Any
future work done with the present method can there-
fore be compared with the studies of Man and Peters
and their co-workers and can thus draw upon the
wealth of information which has been given to the
field of lipid investigation by these authors.
The invaluable technical mistance of Miss Frances Borja is
gratefully acknowledged.
1.
2.
3.
4.
5.
6.
7.
R E F E R E N C E S
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Conference on Biochemical Problem of Lipids, edited
by R. Ruyssen, Brussels, 1956, p. 200.
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6 Suggested by Dr. Liese Abell.
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7. Volume 1
Number 1 MICROTITRATION OF SERUM TOTAL FATTY ACIDS 59
8. Folch, J., M. Lees and G. H. Sloan Stanley. J. Biol.
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