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Potassium, sodium, and calcium are crucial electrolytes of human blood. Among the functions of the electrolytes are maintenance of osmotic pressure and water distribution in the various body fluid compartments, maintenance of the proper pH, regulation of the proper function of the heart and other muscles, involvement in oxidation-reduction or electron transfer reactions, and participation in catalysis as cofactors for enzymes. Therefore, abnormal levels of electrolytes may be either the cause or the consequence of a variety of disorders, and thus the determination of electrolytes in different body fluids in general and in whole human blood particularly is one of the most important functions of the clinical laboratory . Serum or plasma is the usual specimen employed for assay of potassium, sodium, and calcium but not whole human blood. This research intended to create a sensor-array capable of analysing potassium, sodium, and calcium ion-activity in whole human blood based on miniaturised ion-sensors and zero-current direct potentiometry. This research dealt with sensing-side of the sensor-array hoping for integration of these sensors with microelectronics or discrete parts based electronics in due time. Clinics use single electrodes for K+, Na+, and Ca2+ sensing in body fluids. However, single sensing is costly and in emergency cases time consuming as well. Therefore a portable, battery-operated cost- and time-effective sensor array is needed for multi-sensing of r, Na +, and Ca2 + either in emergency cases in field or for patient near bed-side measurement/monitoring. The approach of this thesis to the problem is as follows: This thesis has five phases, all equally important. The first phase, a theoretical phase, mainly deals with the determination of the technique which fits best with chemical sensors and integration with microelectronics, and the decision among many alternative chemicals, whether natural or man-made, available for multi-sensing realisation, by taking into account all the best efforts devoted to development of chemical-sensors. The second phase is the miniaturisation process of the electrochemical sensor-array, either ion-sensors or reference electrode, as much as possible. The third phase is multi-sensing application of the sensorarray for sensing K+, Na+, and ci+ with binary and mixed electrolytes and with artificial and whole human blood respectively, the fourth phase is the sensor-array response with only millilitre or microlitre volumes of whole human blood and the fifth phase is the integration of a sensor array with discrete, parts based, electronic circuitry. So, as a new application a disposable reference electrode has been realised which reasonably fits with the sensor array for the analysis of potassium, sodium, and calcium in whole human blood. Since all the single sensing of these cations have been realised by using a conventional reference electrode, either silver/silver chloride or calomel electrode, which is a bulky and expensive alternative, this research prepared, tested, and implemented a new disposable reference electrode for the sensor-array based on a poly (vinyl alcohol) matrix incorporating a proper amount of Kel. Having a successfully working miniaturised reference electrode, valinomycin, ETH 157, and ETH 1001 ionophores have been used as the selective materials for potassium, sodium, and calcium in whole human blood, respectively. Multisensing applications have only used solutions as testing medium whereas the work described in this thesis goes one step further and uses artificial and whole human blood with fairly reasonable responses.

Although there is information available on the distribution of phospholipids in human serum and red cells, very limited data have been reported for white cells and platelets. To the knowledge of the author, no data have been reported on the distribution of phospholipids among the four blood fractions, of individual subjects. Due to the limited amounts of white cells and platelets present in blood, micromethods are essential for the analyses of these fractions in individual subjects. Although phospholipids have been separated from larger samples by others, their methods were not appropriate for micro amounts. Therefore, procedures were developed in this study which made it possible to isolate and to quantitate the individual components of samples of total phospholipid ranging from 20 to 40 μg. The distributions of phospholipids were determined in serum, red cells, white cells and platelets isolated from the venous blood of four men and four women. The blood fractions were isolated and lipid was extracted by methods previously developed in this laboratory. Total phospholipids were isolated by preparative thin-layer chromatography. Individual phospholipid components were separated and quantitated by the micromethod developed in this investigation. Components were eluted from microchromatoplates and quantitated by analysis of their phosphorus content. This method effectively separated phospholipid samples into lysophosphatidylcholine (LPhC), sphingomyelin (Sph), phosphatidylcholine + phosphatidylserine (PhC + PhS) and phosphatidylethanolamine (PhE). Although all of the fractions contained higher proportional amounts of PhC + PhS than any other component, there were characteristic distributions in each of the blood fractions. Serum was characterized by high PhC + PhS and virtually no PhE. Red cells contained lower amounts of PhC + PhS and more Sph and PhE than any other blood fraction. The distributions of phospholipids in white cells and platelets were similar and resembled the general pattern found in red cells more than that of serum. The marked differences in the distribution of phospholipids among the blood fractions emphasize the importance of concurrent analyses of all blood fractions in studies of human phospholipid metabolism. / Graduation date: 1966

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Although much information is available on lipids in serum and red cells, only limited data have been reported for white cells and platelets. In a recent investigation in this laboratory, the concentrations of cholesterol in the several blood fractions were compared. The high amounts of cholesterol found in white cell-platelet samples together with the wide variability in the proportions of free cholesterol stimulated an interest to study in more detail the lipid composition of the four blood fractions. The concentrations of total lipids, the distributions of lipid classes, and the fatty acid compositions of cholesterol esters, triglycerides, and phospholipids were determined in serum, red cells, white cells, and platelets isolated from venous blood obtained from sixteen adult men and women. Supplementary analyses were made on nine samples of blood from one person at intervals over a four-week period. Modifications of a published method made possible the simultaneous separation of each of the formed elements; serum was obtained in the usual manner. Satisfactory procedures for total lipid extraction from each blood fraction were developed by selective combinations of individual steps of the classical ethanol-diethyl ether and chloroform-methanol methods. The proportional distribution of lipid classes in each blood fraction was quantitated by chemical analyses of cholesterol and lipid phosphorus and subsequent calculations. Lipid classes were isolated by preparative thin-layer chromatography. Methyl esters of the major classes were prepared by direct interesterification and analyzed by gas-liquid chromatography. Concentrations of total lipid in sera and red cells were similar, averaging 603 and 555 mg per 100 ml, respectively. White cells and platelets contained two to three times as much, averaging 3051 and 2587 mg per 100 gm, respectively. The different blood fractions exhibited characteristic distributions of lipid classes. Serum was characterized by high cholesterol esters and triglycerides, together with phospholipids. All cellular components contained higher proportions of phospholipids and free cholesterol than serum. White cells and platelets contained higher amounts of cholesterol esters and free fatty acids than red cells. The fatty acid compositions of cholesterol esters, triglycerides, and phospholipids were not constant among the different blood fractions. The proportions of fatty acids in triglycerides were most similar. Greatest variations in proportions of fatty acids occurred in cholesterol esters. Lipid classes in all cellular fractions were characterized by containing significant amounts of unidentified longer chain fatty acids which were essentially absent in serum. Individual-to-individual variations among the sixteen subjects were greater than day-to-day variations of the one subject for total lipids in serum and red cells, all lipid classes in serum, and phospholipids in all blood fractions. The concentrations of lipid in blood fractions showed no relationship to the age or sex of the subjects or to their assessed intakes of fat. Fat intakes were quite similar, however. Differences in the distribution of lipid classes and fatty acid composition of lipid classes among the four blood fractions for these healthy subjects suggest the desirability of concurrent analyses of all blood fractions in studies of lipid metabolism in humans. / Graduation date: 1965

Graduation date: 1961

The purpose of this study was to investigate the use of excess CO₂ (ExCO₂) as a determinant of the anaerobic threshold (AT) and the subsequent relationship to blood lactate (La). Four highly trained marathon runners (x values, age=30.6 years; % body fat= 8.2; VO₂max = 6 8 ml-kg⁻¹min⁻¹) volunteered to participate in this study. Metabolic and respiratory exchange variables were assessed by an open circuit method utilizing a Beckman metabolic measurement cart interfaced on-line with a Hewlitt Packard 3052A data acquisition system. VO₂max and the treadmill velocity at the threshold of anaerobic metabolism (V[sub=tam]) were obtained from a progressive, treadmill run (.81 kph. > min.) until volitional fatigue. V[sub=tam] (Kilometers per hour, Kph) was calculated from the point of .a non-linear increase in ExCO₂. Subjects performed set treadmill runs of 10 minutes on alternate days. Variations (latin square) included runs at V[sub=tam], V[sub=tam+1], [sub=tam+2], and [sub=tam-1]. Analysis of variance with preplanned orthogonal comparisons and Scheffe post hoc contrasts were used to determine the effects of the treadmill variations on La and ExCO₂. There was no significance found between V[sub=tam] and V[sub=tam-1] for La or ExCO₂. Significance (p < .05) was evident with V[sub=tam]< [sub=tam+1, V[sub=tam]< [sub=tam+2] for La and V[sub=tam]< [sub=tam+2] for ExCO₂. An overall correlation of .89 (p <. 005) demonstrated a high positive relationship between ExCO₂ and La. Findings indicate V[sub=tam] to be a critical point in determining the anaerobic threshold in marathoners, and performance above this demarcation results in a state of anaerobiosis. / Education, Faculty of / Curriculum and Pedagogy (EDCP), Department of / Graduate