1
P-4 8.6 (60 l~g/ml). The free 2.5-OHD was separated from the bound by charcoal adsorption. Using this assay, a detection limit of 0.2 ng 2.5- OHD/tube and a less than 696 non specific binding were obtained. No interference by vitamin D, other vitamin D metabolites, biliary salts, or cholesterol was seen. The recovery of 25-OHD added to serum averaged i0.5% and 100% at 40 and 100 ng/ml respectively. The intra-and inter-assay variation, done in quadruplicate, were 7 and 1096 respectively. The correlation between our assay and that of Haddad and Chyu (1971) (.3. Clin. Endocr. 33, 992) was excellent (r = 0.99, p <0.001). The reference values (nmol-71-+2 s.d.) obtained .from samples of 40 children were 50..5-+21..5 with extremes of 34.8 and 9.5.2. This reliable assay therefore renders the measurement of 2.5- OHD more accessible to clinical chemistry laboratories. I 12 ] STUDY OF THE SEPARATION OF T3, rT3 AND T~ BY REVERSE PHASEHPLC USING UV AND THIN-LAYER ELECTROCHEMICAL DETECTION (TLED) CELL TECHNIQUES. B.R. Hepler, S.G. Weber, B.J. Compton, and W.C. Purdy, Dept. of Chemistry, McGill University, Montreal, Quebec, H3A 2K6. The chromatographic characteristics of T3, rT3 AND T~ were studied as a function of mobile phase composition, pH and temperature using a reverse phase (octadecyl) column. Mobile phases were either n-propanol, methanol or acetonitrile mixed with an aqueous phosphate solution. The pH of the aqueous phase was varied from pH = 2 to pH = 7, and mixed with a given organic at a specific ratio. At a constant aqueous phase pH, the organic phase content was varied from I0-50% (V/V). The influence of temperature was studied dependent upon the organic component from ambient to 70°C. Detection was either by a UV absorbance detector at 254 nm, or by a TLED cell with a glassy carbon working electrode. Results demonstrate that T3, rT3 and T~ can be efficiently separated and eluted within 5-I0 minutes under optimum conditions for each of the solvent systems studied. Both organic mobile phase content and pH variation demonstrated large influences on capacity factor, k', for these hormones. The organic component proved to have the largest influence on k' values both in the context of total content and type. The variation in pH produced changes in k' allowing retention order to be manipulated. Increasing temperature also proved to decrease k' as well as exert a fine tuning effect on chromatographic efficiency. Finally, variation in the pH of the aqueous phase produced the largest effect on efficiency with the best efficiency occurring at low pH values. I 13 I VOLTAMMETRIC IMMI[NOASSAY. S.C. Weber and W.C. Purdy, Dept. of Chemistry, McGill University, Otto Maass Bldg., 805 Sherbrooke St. W., Montreal P.Q. H3A 2K6. The marriage of electrochemical detection and immunoassay is a potentially powerful union. Electrochemical detectors are naturally amenable to flow stream analysis, they have high sensitivity and are inexpensive. Immunoassay possesses specificity and reliability. Using a model system we explored the possibilities of such a marriage, called voltammetric immunoassay (VIA). Consider the labeled molecule (L*) to be an electro- chemically active analog of the hapten (L,analyte). For an efficient assay, one must be able to differentiate between bound L* and free L*. This may be done by making an electro- chemical measurement which is fast with respect to the dissociation of the bound L*. Thus only free L* is measured. This system has been tested using aqueous solutions of codeine (L) with morphine as L*. In other experiments, labelling was carried out with a ferrocene derivative to show the feasibility of electrochemical labelling. In either case, the free L* can be measured in the presence of bound L* using VIA. High backgrounds in urine make the application of VIA to direct analysis in urine difficult. Rather VIA is envisioned as a specific on-line detector for the analysis of certain polypeptides after HPLC separation. I IPHENYTOIN DETERMINATION BY THE SUBSTRATE-LABELED l 14 FLUORESCENT IMMUNOASSAY ($LFIA). R. C. WonB and J. F. Burd, i Miles Labs., Inc., Elkhart, IN 46515, U.S.A. A homogeneous substrate-labeled fluorescent immunoassay to determine the phenytoin concentrations in human serum is de- scribed. A fluorngenlc enzyme substrate, galactosyl-umbelli- fernne, is covalently coupled to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate is non- fluorescent but becomes fluorescent upon hydrolysis catalyzed by bacterial B-galactnsldase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitlve-blnd- ing reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoln concentrations. Using a standard curve, the fluorescence intensity produced by a serum sample can be related to the phenytoin concentra- tion. Results of phenytoin levels in clinical serum samples determined by SLFIA correlated well (correlation coefficient = 0.97) with results obtained by gas chromatographic or en- zyme Immunoassay procedures. The major metabolite of pheny- toin, 5(p-hydroxy)-5-phenylhydantoin, at concentrations ex- pected in serum has no effect on the assay. The SLFIA uses very stable reagents, does not require separation steps, and is rapid and simple to perform. l 15 ~AN ENZYMIC PROCEDURE FOR THE DIRECT DETER- MINATION OF LECITHIN IN AMNIOTIC FLUID. J.D. Artissl, 2 , T.F. Draiseyl, 2 , R.J. Thibertl, z and K.E. Taylor I , IDept. of Chemistry, Univ. of Windsor, Windsor, Ont., N9B 3P4, and 2Depts. of Pathology Salvation Army Grace Hospital and Windsor Western Hospital Centre, Windsor, Ontario. Methods commonly available for the assessment of lecithin concentrations in amniotic fluid, until recently, have involved either thin-layer, or gas- liquid chromatography. These procedures are either time-consuming and expensive or seml-quantitative, at best. One procedure which utilizes enzymes as reagents has been reported, but it too is lengthy and, as with previous procedures, involves an organic solvent extraction. The procedure which we describe is relatively quick, simple, inexpensive, precise and involves no extractions. Preliminary studies suggest that this procedure correlates well with thin-layer L/S ratios yet may be performed in about one-quarter of the time. The enzymatic sequence involves the hydrolysis of choline from lecithin catalyzed by phospholipase D (cabbage). Liberated choline is oxidized to betaine, with the formation of hydrogen peroxide, in the presence of choline oxidase (Arthrobacter globlformis). A red dye is consequently produced by the peroxidase catalyzed coupling of sodium 2-hydroxy-3,5-dichlnrobenzenesulfonate to 4-amino- antipyrene with hydrogen peroxide. The use of this assay in an enzymic procedure for the determination of L/S ratios will also be described. IB |BIOCHEMICALEVALUATIONOF THE NUTRITIONAL AND META- l BOLIC STATUS OF GERIATRIC SUBJECTS. D. Shapcott, S. Hontela, J. Vobecky, D~p. de P~diatrie and D~p. de Sant~ communautaire, Centre hospitalier universitaire de Sherbrooke, Ou~bec, JIH 5N4 and Douglas Hospital Centre, Montreal, Quebec, HdH IR3. This presentation reports part of a study undertaken to evalua- te the nutritional and metabolic status of institutionalised geriatric subjects. A major objective was to determine if dif- ferences existed in this context between those sujbects and a mentally healthy control group of the same sex and age. In 30 female, long-term, hospitalized, psychogeriatric females and 30 mentally healthy female residents of a manor, serum or plasma was analyzed for total protein, cholesterol, triglyceri- des, calcium, magnesium, folic acid, iron and vitamins A, C and E. Chromiumwas measured in urine and in hair. Psychogeriatric patients were found to have significantly lower cholesterol, vitamin A, vitamin C, Ca, higher folic acid and lower hair chromium levels. The effect of brain disorder, institutionalization, psychotropic drugs, lack of physical exercise in some patients, effect of physical disease, insufficient sun exposure, have been proposed as possible causes for metabolic differences. I ]INFLUENCE OF DEXTROSE ON UTILIZATION OF I.V.-LIPID I 17 I.D.Joshi, J.B.Das and A.I.Philippart, Department of Surgery, Children's Hospital of Michigan, Detroit, Michigan 48201. We investigated the influence of Dextrose on the clearance of infused Intralipid. Ten healthy adult dogs were maintained on O.15M NaCI(SAL) or 10% dextrose(DEX) 5ml/kg/hr for 7 hours. Over the mid-4 hours, Intralipid was infused at 0.5g/kg/hr. Serial blood samples were analyzed for chylomicrons (micro- nephelometry), FFA, Sugar and IR-Insulin. The experiments were duplicated with heparin (S0 Units/kg/hr) for the determination

16 Biochemical evaluation of the nutritional and metabolic status of geriatric subjects: D. Shapcott, S. Hontela, J. Vobecky, Dép. de Pédiatrie and Dép. de Santé communautaire,

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8.6 (60 l~g/ml). The free 2.5-OHD was separated from the bound by charcoal adsorption. Using this assay, a detection l imit of 0.2 ng 2.5- OHD/tube and a less than 696 non specific binding were obtained. No interference by vitamin D, other vitamin D metabolites, bil iary salts, or cholesterol was seen. The recovery of 25-OHD added to serum averaged i0.5% and 100% at 40 and 100 ng/ml respectively. The intra-and inter-assay variation, done in quadruplicate, were 7 and 1096 respectively. The correlation between our assay and that of Haddad and Chyu (1971) (.3. Clin. Endocr. 33, 992) was excellent (r = 0.99, p <0.001). The reference values (nmol-71-+2 s.d.) obtained .from samples of 40 children were 50..5-+21..5 with extremes of 34.8 and 9.5.2. This reliable assay therefore renders the measurement of 2.5- OHD more accessible to clinical chemistry laboratories.

I 12 ] STUDY OF THE SEPARATION OF T3, rT3 AND T~ BY REVERSE PHASE HPLC USING UV AND THIN-LAYER ELECTROCHEMICAL DETECTION (TLED) CELL TECHNIQUES. B.R. Hepler, S.G. Weber, B.J. Compton, and W.C. Purdy, Dept. of Chemistry, McGill University, Montreal, Quebec, H3A 2K6.

The chromatographic characteristics of T3, rT3 AND T~ were studied as a function of mobile phase composition, pH and temperature using a reverse phase (octadecyl) column. Mobile phases were either n-propanol, methanol or acetonitr i le mixed with an aqueous phosphate solution. The pH of the aqueous phase was varied from pH = 2 to pH = 7, and mixed with a given organic at a specific rat io. At a constant aqueous phase pH, the organic phase content was varied from I0-50% (V/V). The influence of temperature was studied dependent upon the organic component from ambient to 70°C. Detection was either by a UV absorbance detector at 254 nm, or by a TLED cell with a glassy carbon working electrode.

Results demonstrate that T3, rT3 and T~ can be ef f ic ient ly separated and eluted within 5-I0 minutes under optimum conditions for each of the solvent systems studied. Both organic mobile phase content and pH variation demonstrated large influences on capacity factor, k ' , for these hormones. The organic component proved to have the largest influence on k' values both in the context of total content and type. The variation in pH produced changes in k' allowing retention order to be manipulated. Increasing temperature also proved to decrease k' as well as exert a fine tuning effect on chromatographic efficiency. Finally, variation in the pH of the aqueous phase produced the largest effect on efficiency with the best efficiency occurring at low pH values.

I 13 I VOLTAMMETRIC IMMI[NOASSAY. S.C. Weber and W.C. Purdy, Dept. of Chemistry, McGill University, Otto Maass Bldg., 805 Sherbrooke St. W., Montreal P.Q. H3A 2K6.

The marriage of electrochemical detection and immunoassay is a potentially powerful union. Electrochemical detectors are naturally amenable to flow stream analysis, they have high sensitivity and are inexpensive. Immunoassay possesses specificity and reliability. Using a model system we explored the possibilities of such a marriage, called voltammetric immunoassay (VIA).

Consider the labeled molecule (L*) to be an electro- chemically active analog of the hapten (L,analyte). For an efficient assay, one must be able to differentiate between bound L* and free L*. This may be done by making an electro- chemical measurement which is fast with respect to the dissociation of the bound L*. Thus only free L* is measured.

This system has been tested using aqueous solutions of codeine (L) with morphine as L*. In other experiments, labelling was carried out with a ferrocene derivative to show the feasibility of electrochemical labelling. In either case, the free L* can be measured in the presence of bound L* using VIA.

High backgrounds in urine make the application of VIA to direct analysis in urine difficult. Rather VIA is envisioned as a specific on-line detector for the analysis of certain polypeptides after HPLC separation.

I IPHENYTOIN DETERMINATION BY THE SUBSTRATE-LABELED l

1 4 FLUORESCENT IMMUNOASSAY ($LFIA). R. C. WonB and J. F. Burd,

i

Miles Labs., Inc., Elkhart, IN 46515, U.S.A. A homogeneous substrate-labeled fluorescent immunoassay to

determine the phenytoin concentrations in human serum is de- scribed. A fluorngenlc enzyme substrate, galactosyl-umbelli- fernne, is covalently coupled to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate is non- fluorescent but becomes fluorescent upon hydrolysis catalyzed by bacterial B-galactnsldase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitlve-blnd-

ing reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoln concentrations. Using a standard curve, the fluorescence intensity produced by a serum sample can be related to the phenytoin concentra- tion. Results of phenytoin levels in clinical serum samples determined by SLFIA correlated well (correlation coefficient = 0.97) with results obtained by gas chromatographic or en- zyme Immunoassay procedures. The major metabolite of pheny- toin, 5(p-hydroxy)-5-phenylhydantoin, at concentrations ex- pected in serum has no effect on the assay. The SLFIA uses very stable reagents, does not require separation steps, and is rapid and simple to perform.

l 15 ~AN ENZYMIC PROCEDURE FOR THE DIRECT DETER- MINATION OF LECITHIN IN AMNIOTIC FLUID. J.D. Artissl, 2 , T.F. Draiseyl, 2 , R.J. Thibertl, z and K.E. Taylor I , IDept. of Chemistry, Univ. of Windsor, Windsor, Ont., N9B 3P4, and 2Depts. of Pathology Salvation Army Grace Hospital and Windsor Western Hospital Centre, Windsor, Ontario.

Methods commonly available for the assessment of lecithin concentrations in amniotic fluid, until recently, have involved either thin-layer, or gas- liquid chromatography. These procedures are either time-consuming and expensive or seml-quantitative, at best. One procedure which utilizes enzymes as reagents has been reported, but it too is lengthy and, as with previous procedures, involves an organic solvent extraction.

The procedure which we describe is relatively quick, simple, inexpensive, precise and involves no extractions. Preliminary studies suggest that this procedure correlates well with thin-layer L/S ratios yet may be performed in about one-quarter of the time.

The enzymatic sequence involves the hydrolysis of choline from lecithin catalyzed by phospholipase D (cabbage). Liberated choline is oxidized to betaine, with the formation of hydrogen peroxide, in the presence of choline oxidase (Arthrobacter globlformis). A red dye is consequently produced by the peroxidase catalyzed coupling of sodium 2-hydroxy-3,5-dichlnrobenzenesulfonate to 4-amino- antipyrene with hydrogen peroxide.

The use of this assay in an enzymic procedure for the determination of L/S ratios will also be described.

IB |BIOCHEMICAL EVALUATION OF THE NUTRITIONAL AND META- l

BOLIC STATUS OF GERIATRIC SUBJECTS. D. Shapcott, S. Hontela, J. Vobecky, D~p. de P~diatrie and D~p. de Sant~ communautaire, Centre hospitalier universitaire de Sherbrooke, Ou~bec, JIH 5N4 and Douglas Hospital Centre, Montreal, Quebec, HdH IR3.

This presentation reports part of a study undertaken to evalua- te the nutrit ional and metabolic status of institutionalised geriatr ic subjects. A major objective was to determine i f d i f - ferences existed in this context between those sujbects and a mentally healthy control group of the same sex and age.

In 30 female, long-term, hospitalized, psychogeriatric females and 30 mentally healthy female residents of a manor, serum or plasma was analyzed for total protein, cholesterol, t r ig lycer i - des, calcium, magnesium, fo l ic acid, iron and vitamins A, C and E. Chromium was measured in urine and in hair.

Psychogeriatric patients were found to have signif icantly lower cholesterol, vitamin A, vitamin C, Ca, higher fo l ic acid and lower hair chromium levels.

The effect of brain disorder, inst i tut ional izat ion, psychotropic drugs, lack of physical exercise in some patients, effect of physical disease, insuff icient sun exposure, have been proposed as possible causes for metabolic differences.

I ]INFLUENCE OF DEXTROSE ON UTILIZATION OF I.V.-LIPID I 17

I.D.Joshi, J.B.Das and A.I.Philippart, Department of Surgery, Children's Hospital of Michigan, Detroit, Michigan 48201.

We investigated the influence of Dextrose on the clearance of infused Intralipid. Ten healthy adult dogs were maintained on O.15M NaCI(SAL) or 10% dextrose(DEX) 5ml/kg/hr for 7 hours. Over the mid-4 hours, Intralipid was infused at 0.5g/kg/hr. Serial blood samples were analyzed for chylomicrons (micro- nephelometry), FFA, Sugar and IR-Insulin. The experiments were duplicated with heparin (S0 Units/kg/hr) for the determination