AlllOt PUBLICATIONS

NBS1R 73-178

Microcalorimetric Assay for

Glucose in Human Serum and Plasma

R. N. Goldberg. E. J. Prosen. B. R. Staples. R. N. Boyd, and G. T. Armstrong

Thermochemistry Section

Physical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

April 1973

Interim Report

ared for

' 160 Dnal Institute of General Medical Sciences

,UL5(0 onal Institutes of Health

73*17$ esda ' MarY |and 20014

1913

NBSIR 73-178

MICROCALORIMETRIC ASSAY FOR

GLUCOSE IN HUMAN SERUM AND PLASMA

R. N. Goldberg, E. J. Prosen, B. R. Staples, R. N. Boyd, and G. T. Armstrong

Thermochemistry Section

Physical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

April 1973

Interim Report

This report is to be superseded by a future publication which will receive general distribution and should be cited as a reference.

Please consult the NBS Office of Technical Information and Publications

to obtain the proper citations.

Prepared for National Institute of General Medical Sciences

National Institutes of Health

Bethesda, Maryland 20014

U. S. DEPARTMENT OF COMMERCE, Frederick B. Dent, Secretary

NATIONAL BUREAU OF STANDARDS, Richard W. Roberts, Director

Foreword

The use of clinical laboratory tests of physiological

fluids for specific components has been rapidly increasing in

the past few years. One aspect of this has been an increase

in the number of tests for well-known particular substances

for which satisfactory tests are available. Another equally

important aspect is the increasing need of the physician and

of the research biochemist for ready methods for determining

other substances present in varying amounts in normal and

pathological states. For this reason, the development of new techniques applicable to problems for which optimum solutions

have not been found are of considerable interest in clinical

and biological chemistry.

The universal occurrence of energy changes in chemical

and physiological processes has caused the calorimeter to be

considered as a potential tool for clinical chemical analysis

and for observing biological processes for some years. The

actual applicability, however, has been limited previously by

lack or insufficiency of one or more necessary factors. Such

factors include: instrument sensitivity and operational noise

levels, instruments adapted to use of small samples, simplicity

of instrumental operation and data reduction, and suitable

mechanisms for sorting out specific processes from a melange

of potential interferences. It was the judgment of the authors

and of the National Institute of General Medical Sciences (NIGMS)

that the understanding of these factors had developed to a point

where a well coordinated attack on the application of microcalorimetry would now lead to successful results.

A program was established in the Thermochemistry Section

(Physical Chemistry Division, Institute of Materials Research)

at the National Bureau of Standards, and after some months was augmented with support from the NIGMS to demonstrate the

feasibility of microcalorimetry in some specific areas useful

in the Clinical Chemistry Laboratory. Dr. Robert S. Melville

is the program officer for NIGMS. Collaborative support has

been provided by the Clinical Chemistry Service (Dr. Donald

Young, Chief), and by the Laboratory for Technical Development

(Dr. Robert L. Bowman, Chief, and Dr. Robert L. Berger)

.

This is a report provided at the end of the first year of

the NIGMS support of this work and summarizes a portion of the
work done. This report is one of four covering different aspects

of the NBS Program. For the information of the reader, the titles

and NBS Report numbers of the four reports are as follows:

NBS Report 73-178 Microcalorimetric Assay for Glucose in

Human Serum and Plasma

73-179 Design and Construction of the NBS Clinical

Microcalorimeter

73-180 Testing of the NBS Clinical Microcalorimeter

73-181 Fine Structure in Thermal Growth Patterns

of Bacteria by Microcalorimetry

y " y

" 7-

George T. Armstrong J

Principal Investigator

Edward J. Proven

Project Leader
Microca lor ime trie Assay for Glucose in

Human Serum and Plasma

by

R. N. Goldberg, E. J. Prosen, B. R. Staples

R. N. Boyd, and G„ T. Armstrong

Abstract

Microca lor ime try has been used to perform quantitative assays for

glucose in human serum and plasma samples. The procedure was based upon

the measurement of the heat effect brought about by the following

enzyme catalyzed reaction:

Glucose + ATP"4 He^°kinase

> Glucose-6-Phosphate" 2 + ADP"

3

+ H

+

^

(in buffer)

For this reaction, calorimetric measurements have yielded a value of = -61.4 kJ-mol" 1 in 0.2 M TRIS/TRIS • HC1 buffer (pH = 7.60) at

30.8°C.

Using a procedure that seeks to minimize interfering heat effects

and by application of a calibration curve determined using known

glucose concentrations in water (not serum), fifty-one human serum

and plasma samples have been assayed for glucose. The results cover

a range of 35 to 408 mg glucose per 100 g of serum or plasma and

are in satisfactory agreement with the results obtained using a conventional spectrophotometric clinical procedure. Since our results are based on the assumption of the essential specificity

of enzymes, the agreement of the calorimetric results with the

spectrophotometric results lends strong support to this fundamental

hypothesis of biochemistry.

Table of Contents

Page

1. Introduction 1

2. Enzyme Systems Considered 1

3. Experimental 3

A. Materials 3

B. Apparatus and Procedures 5

4. Results and Discussion 8

A. Glucose/Glucose Oxidase Thermochemistry 8

B. Glucose/Hexokinase Thermochemistry 9

C. Assays on Human Serum and Plasma 9

5 . Acknowledgements 11

6. Appendix 12

7. References 14

Tables

Table I. Summary of Glucose/Glucose Oxidase Results. 15

Table II. Results for the Glucose/Hexokinase System

in Buffer 16

Table III. Summary of Microcalorimetric Assays for

Glucose in Human Plasma and Serum .... 17

Figures

Figure 1. Plot of measured heat effect as function

of u moles of glucose reacted (hexokinase)

reaction) 19

Figure 2. Plot of microcalorimetric (NBS) assay

results for glucose as function of

autoanalyzer (NIH) results 20

1. INTRODUCTION

One may state as a general principle that chemical changes are, essentially

without exception, accompanied by either the liberation to or the absorption

of energy from the surroundings. This energy, in the form of a measurable heat

effect, thus represents a signal, generated by the system itself, that may be

exploited as a quantitative measure of the amount of reaction taking place.

Exploitation of this principle for quantitative analysis is dependent on three

factors: (1) the sensitivity of the calorimetric devices available for use,

(2) the necessary specificity of reaction, and (3) a knowledge of the magnitude

of the heat effect associated with a given quantity of substance and the various

parameters that may affect it.

The first of these three requirements, high sensitivity, is presently

accomplished by means of microcalorimetry [1, 2, 3]. The requirement of

specificity is, in principle, obtainable by the use of an enzyme catalyzed

reaction and is dependent on the availability of a pure enzyme if one is assaying

for its substrate, or conversely, of a pure substrate if one is assaying for the

enzyme. The final requirement, a knowledge of the thermochemistry, is essential

if meaningful measurements are to be made.

This report gives the results of some experiments performed to test the

feasibility of the application of the above principle to clinical chemistry.

Specifically, the objective was to be able to perform quantitative analysis for

glucose in human serum and plasma samples.

2. ENZYME SYSTEMS CONSIDERED

During the course of this work, two different enzymatic reactions were

investigated, namely, those catalyzed by glucose oxidase and hexokinase .

Initial work centered on the use of glucose oxidase , the reaction for which we

write

1
3-D Glucose(buffer) 1 + (^(buffer) Glucose oxidase

>6 _Gluconolactone ( buf f er )

+ H

2 0

2 (buffer) (1)

The gluconolactone will eventually be converted to gluconic acid which in turn

will react with the buffer present. If catalase is present, the hydrogen peroxide

will decompose to water and oxygen:

H

2 02 (buffer) -> H

2 0(buffer) + 1/2 0

2 (buffer) <2)

Several factors may serve to complicate the chemistry. First, it should be noted

that glucose oxidase is essentially specific for 3-D Glucose [4] and thus one is

dependent on the presence of the enzyme mutarotase or self rotation to accomplish the

necessary cv->|3 conversion. Second, the conversion of 6- gluconolactone may require the

presence of the enzyme lactonase [4] to speed up this otherwise slow conversion.

Finally, one must contend with a constraint imposed on the system due to the

limited solubility of oxygen in water, which requires one to insure that the

glucose concentration is always kept below a given level (^18 mg d£ ) . Any

measured heat effect will be attributable to a combination of the several processes

described above, any one of which may serve as a source of experimental difficulty.

The second enzyme considered, hexokinase , catalyzes the following reaction:

Glucose (buffer) + ATP

-4

(buf f er)

Hexo^

nase

> ADP~

3

(buf f er) +

Mg

Glucose-6-Phosphate~ (buffer) + H

^

(3)

The proton produced in the above reaction will in turn react with the buffer

present

:

buffer > buffer'H® (4)

^"Throughout this report, the terminology (buffer) refers to an appropriately

buffered state.

2
The measured heat effect will be the sum of the heat effects associated with

reactions (3) and (4). That reaction (3) goes essentially to completion has

been established by Burton and Krebs [5] who have determined the Gibbs's free energy

change associated with (3) to be -21.3 kJ mol" 1

. This corresponds to an

equilibrium constant of ^5500. Although the chemistry associated with the

hexokinase reaction is simpler than that of the glucose oxidase reaction(s),

hexokinase is known to be not totally specific for glucose in that mannose,

fructose, and D-2-deoxyglucose will be phosphory lated to yield the corresponding

6-phosphates [4]. Nevertheless, this should not be a serious drawback for

purposes of assay of human serum and plasma in that the quantities of these

other carbohydrates are generally small in comparison with the amount of

glucose present [4]. In principle, specificity could be enhanced by use of

the enzyme glucose-6-phosphate dehydrogenase . This is done in the spectrophotome trie

procedure [6] out of the necessity to obtain a satisfactory absorption peak.

However, for the calorimetric (heat measurement) procedure this is not needed,

except perhaps to obtain added specificity.

3. EXPERIMENTAL

2 A. Materials

The reagents used during the course of the experiments were:

Reagents Source

a-D-Glucose, crystalline National Bureau of Standards, Standard

Reference Material No. 916

Adenosine-5 ' -triphosphate , Cal . Biochem, Catalog No* 1191, Lot

disodium salt (ATP) No. 200293 and 001775.

Hexokinase (yeast), lyophilized Cal. Biochem. Catalog No. 376811,

Lot Nos. 020031 and 220069

(Activity ^75 IU/mg)

"Certain commercial equipment, instruments, or materials are identified in

this report to adequately specify the experimental procedure. In no case does such identification imply recommendation or endorsement by the National

Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.

3
Reagents

Hydrochloric Acid (concentrated)

Magnesium dichloride hexahydrate,

crystalline

Potassium dihydrogen phosphate,

crystalline

Sodium hydrogen phosphate,

crystalline

Glucose oxidase , fungal

Catalase , fungal (A. Niger)

Sodium chloride, crystalline

Alkylphenoxy polyethoxy

ethanol (Triton X-100)

Distilled water

Source

Allied Chemical (B&A) Code No. 1090

Allied Chemical (B&A) , no Lot No. given

National Bureau of Standards, Standard

Reference Material No. 186-I-C

National Bureau of Standards, Standard

Reference Material No. 186-II-B

Sigma, Catalog No. G6125, Lot No.

45B-2050 (Activity ~15 IU/mg).

Sigma, Experimental Lot No. 42C-9500

(Activity ^36 IU/mg).

Cal. Biochem, Catalog No. 21926, Lot

No. 71259 (Activity ^15,000 IU/mg)

Sigma, Catalog No. C-40, Lot No.

121C-8010 (Activity vL5,000 IU/mg).

J. T. Baker Chemical Co., Catalog No.

3624, Lot No. 35082

Rohm & Haas Lot No. 5513

National Bureau of Standards

Contaminants of the hexokinase

,

as stated by the supplier, were:

Enzymatic Contaminant

glucose-6-phosphate dehydrogenase

glutathione reductase

phosphoglucose isomerase

myokinase

6-phosphogluconic dehydrogenase

Percent of Total Activity

0.004%

0.001%

0.007%

0.001%

0.001%

Other contaminants in this enzyme preparation included albumin, potassium

phosphate, and magnesium aspartate.

All plasma and serum samples were obtained from the clinical laboratory

(Dr. Donald Young, Chief) at the National Institutes of Health. These

samples were kept frozen at approximately -10°C until ready for use. Samples

were thawed at room temperature (^23°C) a few hours prior to use and inverted

4
many times by hand to mix the sample.

B. Apparatus and Procedures

All calorimetric measurements summarized in this report were

performed using the NBS Clinical Microca lorimeter [3]. The calibration

constant of this instrument, as determined from electrical calibrations,

is 16.70 J v

^ s^ at 30.8°C. The thermopile signal was amplified by a

factor of 10^ using a precision nanovolt amplifier and recorded both in

analog and in digital form. Calculation of the peak areas was accomplished

by numerical integration [3b, 8].

The calorimeter cells were of the type "F" design and made of Kel-F.

Each compartment of a cell holds slightly more than 150 y£ of solution.

Since these cells are not yet disposable, it was necessary to denature any

enzyme remaining in a cell after an experiment by means of a concentrated

urea solution. Cells were then rinsed a minimum of five times with the

buffer being used and sucked nearly dry by means of a vacuum line. Beginning

with run no. 415 (between serum nos . 18 and 19), this procedure was

modified in that a cell, following treatment with concentrated urea, was then

rinsed at least five times with distilled water and air dried completely. The

advantage of this procedure is that it avoids any heats of dilution which may

be caused by traces of water or buffer present on either side of the cell compartment that cause slight mismatch of the solutions in the two cell compartments.

However, by having the cell completely dry one then encounters a difficulty

in mixing due to the thinness of the calorimeter cell and the associated capillary

effect which will not permit the liquid to flow after the hydrophilic

surface treatment wears off. This problem was minimized by tilting a

cell to one side following loading of that side of the cell (the

5
I

compartment for the enzyme solution). Occasionally, however, a mixing problem

did occur. Thus, an inert wetting agent, Triton X-100, was added to the buffer

being used in the preparation of solutions, the volumetric ratio of buffer to

wetting agent being 10^ to 1. To date, no mixing problems have been encountered

using this improved procedure.

Gravimetric procedures were used in the preparation of all solutions.

Weighings were done either on a micicbalance sensitive to ±5 ugm or a semimicrobalance sensitive to ±0.1 mg. All weights given in this report refer to

apparent mass in air.

Measurements of pH were performed on a Corning Model 12 pH meter. The

meter was standardized both before and after any measurements using standard

buffers

.

Interfering heat effects due to a mismatch of the reacting solutions

contained in the respective compartments of a microcalorimeter reaction cell

were minimized by use of two different procedures followed in the preparation

of solutions. The first (method I) was to add a known amount of

the limiting substrate to a given buffered solution containing an excess amount of all other reagents required to carry out the reaction.

This solution was designated as the substrate solution. The enzyme

solution was prepared by addition of the required enzyme (s), preferably

in lyophilized form and free of all contaminants (buffer salts in

particular), to the same buffered solution used in the preparation

of the substrate solutions.

6
The second procedure (method II) that was followed was to prepare first

the substrate solution by addition of a known amount of substrate to a

buffered solution containing excess quantities of all other required reagents.

The enzyme solution was then prepared by addition of the necessary enzyme (s)

again in lyophilized form, to a portion of the substrate solution. Thus,

the substrate contained in the enzyme solution has been pre-reacted. The

reason for the use of this procedure is that in performing assays for glucose

in serum and plasma samples, it is not possible to resort to the first

procedure (method I) described above without having mismatched enzyme and

substrate solutions (in particular, there would be no proteins on one side)

that result in interfering heat effects.

The detailed procedure used for the preparation of solutions for assays

of glucose in serum and plasma was as follows:

1. Buffer Solution . To a vial, containing 220 mg of the disodium salt

of ATP and 30 mg of MgCl

2 «6H2 0, add 1.0 ml of 2 . 0M TRIS /TRIS -HC1 buffer,

pH = 7.60. Mix to dissolve all solids and store this solution at approximately 4°C.

2. Substrate Solution . To 1.0 ml of a serum (or plasma) sample to be

assayed for glucose, add 0.1 ml of the solution prepared in (1) above. Mix

thoroughly. The amount of dilution of the serum (or plasma) sample must be

known quantitatively.

3. Enzyme Solution . To at least 100 yg of lyophilized hexokinase , add

400 ul of the substrate solution. Mix thoroughly.
I

I

The above solutions should suffice for at least three calorimetric measurements for glucose in serum.

To illustrate the nature of this analysis, a sample experiment showing

raw data and calculation procedures has been included in the Appendix to

this report.

As is the case with most measurements, and in particular those for which

a high state of reliability has not yet been achieved, it was necessary to

drop the results of some calorimetric experiments. Results were dropped

for several legitimate reasons that included leaks, the most frequent cause,

premixing of the solutions in the two reaction cell compartments, as

evidenced by excessively low values for the measured heat, lack of sufficient

enzymatic activity, inability of the solutions in a reaction vessel to mix,

and miscellaneous operator errors such as failure to obtain a complete record

of the data.

4. RESULTS AND DISCUSSION

A. Glucose in Water /Glucose Oxidase runs

A summary of results for the glucose oxidase system is shown in

Table I. As is evident from these results, some experimental factor

was not under control. Since the hexokinase system seemed more promising,

we went on to study that system rather than trying to improve the

glucose oxidase system.

8
B. Glucose in water /Hexokinase runs.

Numerical results for the glucose/hexokinase system in buffer (not in

serum) are given in Table II. A plot of these results is shown in figure 1. A

molar energy of reaction (ZH) equal to -61.4 kJ mol ^ was obtained from a least

squares fit to this data. This measured energy is the sum of the energies

associated with processes (3) and (4) described in Section 2. The energy

of buffer protonation, process (4), has been reported [9] to be -47.5 kJ mol

^

and represents 78 percent of the measured exothermic heat effect. Thus, the

molar energy of reaction for process (3) , the phosphorylation of glucose-6-

phosphate by ATP, is -13.9 kJ mol \ A check on this value could be obtained

by measurement of the energy of reaction in a second buffer system with a

corresponding correction for the heat of protonation of that buffer. This

has not been done yet.

C. Assays Performed on Human Serum and Plasma Samples

The results of microcalorimetric assays for glucose in human serum

and plasma samples are given in Table III. In most cases, calorimetric

measurements were performed in triplicate. The results of these individual

measurements, expressed in terms of mg glucose per 100 g of serum or plasma,

are given in column three. The average of these determinations are presented

in column four, the expressed uncertainty being the average deviation. In

column five are given the results of glucose determinations performed on

these same samples at the clinical laboratory of the National Institutes of

Health on an autoanalyzer using the enzyme glucose oxidase . Numbers in this

column that are not in parentheses were obtained chronologically prior to the

microcalorimetric measurements, while those in parentheses were obtained after

the calorimetric measurements had been performed. This was done to test for
any possible changes in glucose concentration with time or our handling of

the serum samples. Since the autoanalyzer results are expressed in units of

mg glucose per d& of serum or plasma, a rigorous comparison of these results

would require a knowledge of the density of the individual samples. This

is expected to change the values by only a few percent. Nevertheless, it

should be noted that the mean of the fifty-one calorimetric analyses

(110.0 mg glucose per 100 g serum or plasma) is in good agreement with

the mean of the autoanalyzer results (110.4 mg glucose per d& serum or

plasma)

.

A plot of the microcalorimetrically determined glucose concentration

(units of mg glucose per 100 g serum or plasma) as a function of the glucose

concentration (mg glucose per dl serum or plasma) determined by NIH using the conventional glucose oxidase method with an autoanalyzer is shown in figure 2. The

plot covers a range from 35 to 408 mg glucose per 100 g serum (plasma).

The mean of the absolute deviations between the two sets of numbers is

6.7 mg glucose per d& of serum (plasma), assuming a density of unity for the

samples

.

Several factors still remain to be investigated before the spectrophotometric method as applied to the quantitative determination of glucose

is fully satisfactory. These factors include:

(1) Investigation into possible causes of the several large discrepancies between the calorimetric and spectrophotometry results.

(2) A study of several possible interferences, such as the effect of pH,

addition of mannose, fructose, etc.

(3) An understanding of the cause of and a better control of the endothermic

heat effect observed when serum or plasma is mixed with itself ^ and the

endothermic remixing energy (see appendix).

It is worthwhile to point out that the closeness of the agreement of the

results obtained between the calorimetric and spectrophotometry methods may have bee

fortuitous, in the sense that both methods, and in particular the newer microcalorimetric method, may have associated with it several possible sources of

systematic error that have not yet been recognized. Nevertheless, the results

l n
do serve to indicate the essential validity of the calorimetric approach, as

performed in a research laboratory, to the assay of glucose in human serum

and plasma samples. It should also be noted that since the basis of the

measurement technique rests on the assumption of the essential specificity

of enzymes, the results do lend strong support to the correctness of this

basic hypothesis of biochemistry. It should also be pointed out that the

calibration curves used were performed on glucose in water (not in serum)

and still gave reasonably correct values for glucose in serum.

5. ACKNOWLEDGEMENTS

We wish to thank Drs . Sam Margolis and Robert Schaffer of the Organic

Chemistry Section (NBS) and Dr. Jo Everse of the Department of Chemistry

at the University of San Diego for their helpful discussions pertaining to

the biochemistry pertinent to this investigation. We also thank Dr. Donald

Young for supplying serum and plasma samples, as well as the results of

glucose analyses for these samples performed in the clinical laboratory

of the National Institutes of Health, and Dr. Robert L. Berger of the

Laboratory for Technical Development, Heart and Lung Institute, National

Institutes of Health, for his assistance and encouragement in this project.

11
6. APPENDIX - MICROCALORIMETRIC ASSAY FOR GLUCOSE (Sample No. 6211)

Raw Data

Te 1.02278 § ef strum (sample no. 6211) was added 0.10827 g ©f buffered

solution containing ATP and magnesium chloride. This selutlen was mixed

thoroughly and is the substrate solution described previously in this

report (see Section 3.B). The enzyme solution was prepared by addition of

0.51599 g of substrate solution to 277 yg of hexokinase .

The mlcrocalorimeter cell was then loaded with 0.11030 g of enzyme

solution and 0.15405 g of substrate solution into the respective cell

compartments. An area of +2.7821 millivolt-seconds (mvs) was obtained

for the first mixing, while the remix gave an area of -0.399 mvs.

Treatment of Data

The heat effects to be associated with the first and second mixings are,

respectively

,

Q = (16.7 mJ»mv

-1

.s _1

) (+2.7821 mvs) = +46.461 mJ (exothermic)

Q

2 = (16.7 mJ.mv

-1

-s -1

) (-0.0399 mvs) = -0.666 mJ (endothermic)

It should be noted that this endothermic heat effect, +0.666 mJ, in this specific

instance, was not observed in glucose in water determination. The

origin of this effect is not yet known and constitutes a source of ambiguity

in the data treatment on the order of a few percent in the case of the glucose

determinations. If it is assumed that this effect is a thermal interference,

characteristic of some property of serum or plasma, one can reason that the

heat effect legitimately to be associated with the glucose/hexokinase reactions

is equal to (Q - Q ;) or +46.461 - (-0.666) = +47.127 mJ in this instance. It

should be emphasized that at present, the cause of this endothermic effect is

not yet known and that some other treatment of the data, e.g. taking (Q^ + Q^)

12
or just alone, might be the more accurate procedure. Nevertheless, for

purposes of this report, the procedure of exclusively taking (Q - 0^) was used

to obtain the results of all glucose analyses reported.

Using our measured heat effect of +47.127 mJ and a knowledge of the

equation of the calibration curve of the glucose /hexokinase reaction (see fig. 1), we

calculate that 0.730 y moles of glucose have been reacted in 0.15405 g of

substrate solution. Applying a correction for the dilution of the serum

by the buffered solution containing ATP and magnesium chloride, one can

calculate the concentration of glucose in the serum sample:

glucose concentration (y moles per g serum) = 0.730

0.15405 x

( 1.02278 + 0.10827)

1.02278

5.240.

This corresponds to a 94. ^ mg of glucose per 100 g of serum. The spectrophotome trie

determination performed at the National Institutes of Health yielded

a value of 93 mg of glucose per dH of serum.

13
7 . References

1. "Biochemical Microcalorimetry , " H. D. Brown, editor, Academic Press,

New York, 1969.

2. T. H. Benzinger in "A Laboratory Manual of Analytical Methods of Protein

Chemistry," P. Alexander and H. P. Lundgren editors, Pergammon Press,

Oxford and New York, 1969, page 93.

3a. E. J. Prosen, " Design and Construction of the NBS Clinical Microcalorimeter",

NBS Report No. 73-179 ,(1973).

3b. E. J. Prosen and R. N. Goldberg, "Testing of the NBS Clinical

Microcalorimeter", NBS Report 73-180, (1973).

4. "Biochemists Handbook," Cyril Long editor, D. Van Nostrand Company Inc.,

Princeton, 1961.

5. K. Burton and H. Krebs , Biochemic J., _54, 94 (1953).

6. "Fundamentals of Clinical Chemistry," Norbert W. Tistz editor, W. B.

Saunders Company, Philadelphia, 1970.

7. "Handbook of Biochemistry," H. A. Sober editor, The Chemical Rubber Co.,

Cleveland, Ohio, 1968.

8. R. N. Goldberg, R. L. Nuttall, E. J. Prosen, and A. P. Brunetti, Digital

Data Acquisition and Computer Computation Applied to Calorimetric Experiments, NBS Report No. 10437 (1971).

9. G. Ojelund and I. Wadso', Acta Chemica Scand. , 22, 2691 (1968).

10. J. M. Sturtevant, J. Phys . Chem. 45, 127 (1941).

11. M. A. Kabayama, D. Patterson, and L. Piche, Can. J. Chem. J36, 557 (1958).

14
Table I

Run No.

Summary of Glucose/Glucose Oxidase Results

u moles of glucose reacted Q(mJ) AH(kJ mo

82 0.0890 16.65 187

83 0.0891 9.03 101

84 0.0917 10.65 116

85 Zero 0.45 —

116 0.0826 8.41 102

117 0.0854 17. 72 207

119 0.0861 16.90 196

120 0.0845 15.55 184

,-1,

Average = (156 ± avg. dev. 43) kJ mol

Procedure used for preparation of Solution : Method I

Buffer : 1/15 M S^rensen's phosphate [7] containing 0.146 gm of sodium

chloride per liter, pH = 6.0.

Glucose Concentrations : 5.86 x 10 ^ mol (g soln) ^ in runs no. 82 to 85, or 10.6 mg d£ _1

.

5.47 x 10 ^ mol (g soln) in runs no. 116 to 120, or 9.9 mg dJT 1

.

Enzymes : Sigma Glucose Oxidase (Lot No. 45B2030) and Catalase (Lot No.

121C-810) were used in runs no. 82 to 85.

Sigma Glucose Oxidase (Lot No. 42C-9500) and Cal Biochem Catalase

(Lot No. 71259) were used in runs no. 116 to 120.

In all cases the enzyme activity should have been sufficient to bring

about complete reaction within a few seconds.

-1

15
Table II

Results for the Glucose/Hexokinase System in Buffer

u moles of Q (mJ) Q . (mJ)

Difference

Run No. j meas calc Q(meas)-Q(calc), A Difference

mJ

176 0.00 3.25 2.31 +0.94 -40.7

177 0.00 1.88 2.31 -0.43 +18.6

178 0.00 2.18 2.31 -0.13 + 5.6

179 0.3849 25.18 25.94 -0.76 + 3.0

180 0.3797 25.89 25.62 +0.27 - 1.1

181 0.3860 25. 33 26.01 -0.68 + 2.6

173 0.7503 48.39 48.37 +0.02 - 0.0

174 0.7582 50.40 48.86 +1.54 - 3.0

175 0.7529 48.03 48.53 -0.50 + 1.1

182 1.1193 70.30 71.03 -0.73 + 1.0

184 1.0954 71.66 69.56 +2.10 - 3.0

185 1.1146 69.08 70.74 -1.66 + 2.4

Procedure used for preparation of solutions : Method II

Buffer:

Glucose

0.2 M TRIS/TRIS

Concentrations

•HC1, pH = 7, 0.00 u moles.60

glucose (g soln) in runs no. 176 to 178,

0.0 mg d£ .

2.583 y moles glucose (g soln) ^ in runs no. 179 to 181, or 46.5 mg dl .

5.013 y moles glucose (g soln) ^ in runs no. 173 to 175, or 90.3 mg diT 1

.

7.492 y moles glucose (g soln) in runs no. 182 to 185, or 135.0 mg diT 1

.

Enzyme: Hexokinase (Cal Biochem Lot No. 020031).

In all cases, the enzyme activity should have been adequate to bring complete

reaction within 10 seconds.

*Values given under the heading Q ca^ c were obtained from a least squares fit of

the experimental data.

16
Summary of Microcalorimetric

Table III

Assays for Glucose in Human Plasma and Serum

NBS Micro calorimeter NIH Autoanalyzer

PIIT X doo cm o Hid (Glucose Hexokinase) fGlnrnciP Dyi riasp) Dif f erence

No. No. mg glucose/100 g plasma Average mg glucose/d£ plasma NBS-NIH

Xi 1U1 - 1U5 - 1UJ 103±2 103 0

2 105 167 - 175 - 168 170±3 162 +8

3 115 231 - 239 - 233 234 + 3 224 +1U

4 111 81 - 76-76 78±3 87 -9

5 113 57 - 59 - 64 60±3 67 -7

6 108 189 - 199 194±5 207 -13

NBS Microcalorimeter NIH Autoanalyzer

Serum (Glucose Hexokinase) (Glucose Oxidase) Difference

No

.

No

.

mg glucose/100 g serum Average ttip pi ucose /d£ serum NBS-NIH

1 30208 91 - 90 - 91 91±0.3 90 +1

2 30219 76 - 71 - 74 74±2 73 +1

3 31206 63 - 61 - 60 61±0.8 72 -11

4 1274 188 - 189 - 192 19011 170 +20

5 2003 207 - 199 - 196 201±4 194 +7

6 2008 66 - 68 - 69 6o±l 70 -2

7 2012 62 - 62 - 66 64±2 67 -3

« 901 £m \J -L 7J 93 - 89 91±2 88 +3

q 900Q 73 - 77 - 88 79±6 79 o

i n 9009 95 - 100-97 98±2 9 3 +5

1

1

X X X1 Jl11 6VJ 342 - 351 346±5 330 +16

A-£- 19 761 U 65 - 65 - 69 66±2 67 -1

1J 909 7 69 - 65 67±2 66 +1

14 9m n 91 - 83 87±4 90 -3

X 3 Z.U ox 86 - 83 85±1 85 0

16 5237 63 - 56 - 60 60±2 67

"7 - /

17 5263 81 - 79 - 79 79±1 75 +4

18 5216 94 - 95 94±0.4 87 +7

19 9279 102 - 104 - 104 103+1 96(89) +10

20 9216 411 - 406 - 408 408±2 378(365') +26

17
NBS Microcalorimeter NIH Autoanalyzer

No.

Serum

No.

(Glucose Hexokinase)

mg glucose/100 g serum Average

(Glucose Oxidase)

mg glucose /d£ serum

Difference

NBS-NIH

21 5233 Q 9 y z OO Q 9 yu y z 91±1 87 +4

22 5234 7Q/y 7 Q /is 78±0.5 82 -4

23 5235 £9OZ DJ — DO 64±2 66 -2

24 5253 zoy 9 Q A OQ£ zyo — zyo 294±3 318 -24

25 6211 Q A y^ 94 93 +1

26 6247 Qy j

Q Q y o 97±2 91 +6

27 6248 £A £ 7 D / 66±1 77 -11

28 6237 64 - 59 - 64 63±2 63 0

29 6253 70 - 68 - 74 71±2 75 -4

30 9219 ±10 11 J llo 116±2 122(108) +1

31 9243 79J Z 67 69±3 79(70) -6

32 9310 71 AD 75 - 70 74±2 83(70) -3

33 10392 1 AA-•145-148 146±1 152(143) -1

34 9251 36 - 36 35±1 42(38) -5

35 10364 1 9 A 125 - 130 127±2 116(81) +28

36 10401 1 AA 141 - 147 145±2 150(137) +1

37 6272 J o 62 - 60 60±1 64(65) -4

38 6276 90 92 - 88 90±2 92(93) -2

39 23288 105 106 - 108 106±1 115(110) -7

40 24327 csy 86 - 87 87±1 93(90) -5

41 6286 7 1 74 74±0.5 83(80) -7

42 23292 11U 114 - 114 113±2 125(124) -12

43 23265 70 68 - 69 69±0.6 80(81) -11

44 23266 68 67 - 67 68±0.6 80(81) -12

45 23275 67 66 - 65 66±0.4 75(75) -9

Average for 51 samples = 110.0 110.4 -0.4

(Mean Avg . Dev . = 1.9)

See discussion in text for explanation of various aspects of this table.

18
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form NBS-114A (1-71) U.S. dept. of comm. 1 . PU B LIC AT ION OR RE PORT NO. 2. Gov't Accession

BIBLIOGRAPHIC DATA „„ „ _„

No. SHEET NBSIR 73-178

3. Recipient's Accession No.

4. TITLE AND SUBTITLE

Ml ClTOCa 1 Or imP tv\ r AQaav -For- P 1 1 , /> no a 4 n Unman

Serum and Plasma

5. Publication Date April 1973

6. Performing Organization Code

7. author(s) Ro N. Goldberg, E. J. Prosen, B. R, Staples,

R. N. Boyd, and G. To Armstrong

8. Performing Organization

NBSIR 73-178

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DFPARTMFNT OF rflMMFRTF

WASHINGTON, D.C. 20234

10. Project/Task/Work Unit No. 3160411 and 3160118

11. Contract/Grant No.

12. Sponsoring Organization Name and Address

National Institutes of Health

ue trie soa, rjaryianu zuujlh13. Type of Report & Period

Covered

Interim

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

6. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.) Microcalorimetry has been used to perform quantitative assays for glucose in

human serum and plasma samples. The procedure was based upon the measurement of the

heat effect brought about by the following enzyme catalyzed reaction:

Glucose + ATP"

4 He*°kimse

> Glucose-6-Phosphate'

2 + ADP

-3

+ H

+

(in buffer)

Mg

For this reaction, calorimetric measurements have yielded a value of AQ = -61.4

kJ-mol" 1 in 0.2 M TRIS/TRIS -HCl buffer (pH = 7.60) at 30o8°C.

Using a procedure that seeks to minimize interfering heat effects and by

application of a calibration curve determined using known glucose concentrations in

water (not serum), fifty-one human serum and plasma samples have been assayed for

glucose. The results cover a range of 35 to 408 mg glucose per 100 g of serum or plasma and are in satisfactory agreement with the results obtained using a conventional

spectrophotometry clinical procedure. Since our results are based on the assumption • of the essential specificity of enzymes, the agreement of the calorimetric results

with the spectrophotometric results lends strong support to this fundamental

hypothesis of biochemistry.

17. KEY WORDS (Alphabetical order, separated by semicolons^nalysis ; biochemical microca lorime try ; clinical microcalorimetry; clinical chemistry; enzyme specific reactions; glucose in

serum; hexokinase : microcalorimetry; serum.

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[^UNLIMITED.

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