Measurement of Plasma Cholesterol Concentrations Glucose

Measurementof Plasma Cholesterol Concentrations Glucose

Cholesterolis one of the compounds that is normally found in blood plasma of allorganisms. Other components of blood plasma include glucose, proteinsand lipids. Cholesterol has been found to be a steroid with asecondary hydroxyl group attached to the third carbon within a lipidmolecule. Its synthesis normally occurs in different tissues rangingfrom the liver to intestinal walls. Studies have revealed that 75% ofthe entire cholesterol in blood plasma is synthesized within thetissues while only 25% comes from the daily dietary intake.605 to 80%of this cholesterol is esterified and about 75% is normallytransported within the body by low-density lipoproteins during therest by high-density lipoproteins. High cholesterol levels in bloodplasma have been attributed to metabolic diseases likehypercholesterolemia. Low levels of cholesterol have also beenobserved in disorders that like hyperthyroidism and malabsorption ofnutrients within the blood cells

Bloodcholesterol has since the past been determined in serum and mostcases its concentration in serum and that of circulating plasma arealways considered to be the same. Anticoagulants are normally addedto separate the water from the serum and hence analysis mostly byspectrophotometry. A scientist by the name Schoenhermer in hisanalysis of cholesterol in blood plasma reported that oxalated bloodhad low cholesterol content compared to serum and heparinized blood.Other researchers also done in the same field revealed almost sameresults.

Ina study to determine total cholesterol levels in hypercholesterolemiapatients of 38 human blood samples and normal 8 dogs’ blood samplesusing the method of a bell, et al. revealed that cholesteroldifference between serum and plasma was proportional to serumconcentration that was the cholesterol levels. The results alsoindicate that the effects of anticoagulants heparin, sodium chlorideand EDTA increased when their quantities within the plasma increased(Smith et al. 73).

Reportsfrom egg nutrition council revealed that according to Australiahealth survey on blood cholesterol levels, one out of every threeAustralians aged between 18years and above, that is 32.8% of thetotal population of about 5.6 million has abnormal or high levels ofcholesterol in their blood. The worrying trend was that only 10% ofthese people were aware of their situation indicating that mostpeople did not look at it as a long-term problem (Egg NutritionCouncil). Since high plasma cholesterol levels pose a high risk forheart disease development, it was important to study the causes ofthese cholesterol levels in Australians blood that was attributed tothe dietary components of their food which contained most lipidshence solve the problem. The sources of dietary cholesterol studiesindicated that eggs contributed to 100mg/day consumption in everyperson that consumed 3 to 4 eggs per week. The results also indicatedthat there were hype respondents who their cholesterol levelsincreased with intake of eggs by 100mml/L and hence contributed tohigh cholesterol levels.

Studiesconducted by Francisco Grande, et al on cholesterol measurement inserum and in plasma, revealed that anticoagulants present in bloodplasma contributed to decreasing in cholesterol level in the plasma.This could be explained by the changes in water concentration withinthe cells and plasma as a result of changes in hematocrit that isnormally produced by the coagulants. A study conducted by HiroshimaUniversity school of Medicine investigated the Effects of dietarycholesterol as well as fatty acids on plasma cholesterol level, andhepatic lipoprotein metabolism. The study on animals fed on 0.1% dietcomposed of cholesterol for 2 weeks revealed that dietary linoleicacid increased the effects of cholesterol very low-densitylipoprotein cholesterol secretion from hepatocytes. This researchalso revealed a close relationship that exists between plasmacholesterol levels and occurrence of coronary heart diseases andrecommended reduction of cholesterol levels to reduce risk ofcontraction of atherosclerotic diseases. This was mostly associatedby consumption of foods rich in saturated and polyunsaturated fattyacids that increase the cholesterol levels in blood plasma (Ohtani etal. 1413).

Acohort study was conducted to examine the relationship between plasmacholesterol concentration and mortality from major causes of deathwithin the civil service offices in London, England in 1967 to 1969.The findings revealed that in a population of 17718, 4022 deathsoccurred during the 18years of study and out of this, the totalmortality increased with cholesterol level with the highest onesbeing contributed by coronary diseases. Mortality in small groupswas, however, non-significant compared to the larger groups. Healthstate and socioeconomic conditions at the time of study alsocontributed to the increased cholesterol levels hence coronarymortalities. High blood cholesterol levels have been identified byvarious researches to be the principal contributing factor of heartdisease and cellular diseases like atherosclerosis among otherfactors (Ehrlich).

Thisexperiment has been designed to determine the concentrations ofcholesterol in blood plasma of sampled student. This researchmeasured the levels of cholesterol in the blood sample and alsocompared these levels to the standard levels in a normal human beingto establish the risk of hypercholesterolemia in the subject blood.The blood was sampled from students and centrifuged then run throughthe spectrophotometer to determine its absorbance and hence estimatethe cholesterol levels.

Thisexperiment is important in that it will create awareness of the riskof high cholesterol levels of an individual and also helping todetermine the type of food that is required for a normal person henceprevent hypercholesterosis and consume a diet that has lesscholesterol.

Themain objective of this experiment was to determine the concentrationof cholesterol in blood plasma of the subject under study. It alsoaimed at comparing the cholesterol levels obtained with that of thenormal or abnormal standard or levels of a healthy human being whichare normally associated with specific diseases within the states. Forexample, hypoglycemia associated with high glucose levels in bloodplasma, hypercholesterolemia associated with high cholesterol levelsin blood and hypoproteinemia associated with high protein levels inthe blood. This was determined using spectrophotometer and beers lawbased on the graphical representation of the absorbance levelsmeasured in nm.

Theproblem of high or low cholesterol levels is the source of thisexperiment since it will help in identifying the dietary requirementshence reduce the effects associated with high or low cholesterollevels in blood plasma.This experiment hypothesized that the subjectunder study will have normal cholesterol levels in his or her bloodplasma. This experiment further seeks to establish the serumcholesterol level of the subject under study. Just like studies haveshown that coronary heart disease mortality increased with increasein blood plasma cholesterol level. The amount of cholesterol producedby the human body is enough for metabolic processes. The additionalcholesterol levels from dietary intake are believed to be the solecontributor to these diseases. The normal range of cholesterolconcentration in the human being has been found to be 130 to250mg/dl.

Methods

Ablood sample was obtained from the subject or student using a lancetby first cleaning one finger with alcohol pad squeezing the fingerand pricking with a lancet to allow collection of the blood samplewith a capillary tube approximately 50uL/capillary. Plasma was thenisolated from the blood sample by centrifuging for 5min in acentrifuge machine. After which the serum was separated from bloodplasma and put in the respective test tube.

Thereagents were then added to the components and put in theirrespective test tubes as follows the water, standard, plasma, normaland abnormal serum then absorbance for each was measured andrecorded. Five test tubes were obtained and labeled as (U) Unknown, Sstandard, B blank, Normal and abnormal serum. Using a mechanicalpipette, each 5ml of the cholesterol reagent was pipette into each ofthe tubes. Using different micropipettes, 50uL of serum was added totest tube labeled U, 50uL of the standard into one labeled S and 50uLof water into one labeled B. 50Ul was also added into a test tube ofnormal and abnormal serum respectively. Each tube with 5.05ml of thesolution was then tapped gently to mix and allowed to stand at roomtemperature (around 25 degrees centigrade) for 10 minutes. Thesolutions were then transferred to five cuvettes and spectrometerstandardized at 500nm using the blank solution B. Absorbance of thesolutions S and U, Normal and Abnormal Serum were measured andrecorded. Using Beers law formula, the concentration of cholesterolin unknown plasma sample was calculated. A graph of absorbance valuesfor S vs. Cholesterols concentration in mg/dl was also plotted andused to determine the unknown cholesterol concentration U.

Table1: Reagents mixture used in spectrometer analysis

Tube

Serum

Standard

Water

Reagent

Unknown (U)

50uL

5ml

Standard 1 (S) 200mg/dl

50uL

5ml

Blank (B)

50uL

5ml

Normal serum

50uL

5Ml

Abnormal serum

50uL

5ml

Cplasma = A plasma/A standard x C standard where A is from absorbancecurve.

Alldirections from the lab were followed as per the outlined procedureBIOSC 140 Human Physiology lab. No protocol was deviated startingfrom collection of the blood samples to the final process ofdisposing of the used serum. All safety precautions were alsofollowed during the experiment with no significant change experiencedin the protocol during the experiment. The manual used to carry outthis experiment was entitled BIOSC-140 Human physiology lab 03measurements of plasma concentration Fox 2.1, Glucose, cholesteroland protein.

Results

Table2: Measurements of plasma cholesterol concentration

Variables

Absorbance values

Concentrations mg/dl

Blank water

0.00

0

Standard 1

0.185

200

Unknown Blood

0.16

172.9

Normal Serum

0.14

151

Abnormal Serum

0,21

227

Table3: Concentrations of reagents from beers law

Variables

Beers Law

Unknown Blood

173

Normal Serum

175

Abnormal Serum

262.5

Cx= Cstnd= Ax/Astnd = 200x 0.16/0.185 = 173

Table3 shows the calculated concentrations of unknown blood, normal serumand abnormal serum using beers law. Which states that?

Absorbanceand Concentration Are Directly Proportional

Beer’sLaw:

Cx= Cstnd = Ax/Astnd where

C= concentration

x=unknown

std=standard

Standardconcentrations for glucose have been found to be 100mg/dl,Cholesterol 200mg/dl and that of protein is 2, 4, 6, 8, to 10g/dl.

Figure1: Graph of Absorbance values vs. Cholesterols concentration in mg/dl

Thegraph indicates that the concentration of unknown blood was 173 atabsorbance 0.185. Normal serum was 151 while abnormal serum 227mg/dl.The trend indicates that concentration of unknown blood is higherthan that of normal and abnormal serum (Figure 1).

Discussion

Theexpected results for the concentration of cholesterol in blood plasmaof the subject was between 130 to 250mg/dl that in most cases is thelevel of a normal human being. The results obtained indicated acholesterol concentration of 172.9mg/dl (Table 2) which was withinthe expected range. The calculated concentration of cholesterol asper Beers law was obtained as 173mg/dl that showed that the subjectcholesterol levels were within range hence he had normal cholesterollevels. The hypothesis that was the subject had normal cholesterolrange was hence positive. This indicates that the experiment wassuccessful. In line with this, the beer law also confirmed that thescientifically calculated cholesterol concentration was also withinrange (Figure 1).

Normaland abnormal cholesterol concentrations within the blood serum alsoshowed that the serums were of a normal person. The concentration ofcholesterol present in blood plasma can therefore only be testedusing laboratory tests, and hence the experiment proves this. Theabnormal cholesterol levels are also critically significant in thatthey help to test the person’s risk of contracting and developingcoronary dieses. It also helps in ensuring that the subject restrictshimself to the recommended dietary foods that are free ofcholesterol. The human body requires cholesterol, but too much in thebody is very dangerous especially when it comes to heart dieses. Thebody is also capable of manufacturing enough cholesterol that itrequires for cellular functions (Edington et al. 333).

Theresults obtained hence matched the expected results and hence apositive hypothesis obtained. The possible sources of error that maybe reduced to achieve more accurate results may be in measuring theexact quantities of the reagents, ensuring that the conditions oftemperature are maintained as required since high temperatures tendto denature proteins and enzymes. Also, centrifugation to be done ata suitable cycle since too much time if the sample is left tocentrifuge may damage the cholesterol structure. The main objectiveof this experiment that was to determine the concentration levels ofcholesterol in the subject blood plasma and compare it with thenormal level of a normal human being blood was hence achieved.

Conclusion

Theobjectives of the experiment were all achieved, and the cholesterollevel in the subject blood plasma was determined to be 173mg/dl. Thestudy objective, which was to determine the concentration ofcholesterol in blood plasma, compare the cholesterol levels obtainedwith that of the normal or abnormal standard or levels of a healthyhuman being, and the effects of cholesterol on a healthy human wererealized. Therefore, the experiment can be regarded as successful.

WorksCited

Ehrlich,Steven. Hypercholesterolemia.University of Maryland Medical Center, 27 Mar. 2015. Web. 3 Nov.2015.

EggNutrition Council. Eggs,Plasma Cholesterol and Lipoproteins.Egg Nutrition Council, 2014. Web. 3Nov. 2015.

Edington,Jacqueline, Moira Geekie, and Robin Carter et al. Effect of dietarycholesterol on plasma cholesterol concentration in subjects followingreduced fat, high fibre diet. BritishMedical Journal294(1987):333-336.

Ohtani,Hiromasa., Kozo Hayashi, Yasuhiko Hirata, and Shinya Dojo et al.Effects of dietary cholesterol and fatty acids on plasma cholesterollevel and hepatic lipoprotein metabolism. Journalof Lipid Research,31 (1990):1413-1422.

Smith,George., Martin Shipley, Michael Marmot, and Geoffrey Rose. PlasmaCholesterol Concentration and Mortality: TheWhitehall Study. The Journal of the American Medical Association,267, 1 (1992): 70-76.