ISEC User's Manual

(working version) Version 3.4a Roman Hovorka September 1994 READ FIRST ISEC, which stands for "I(nsulin-)SEC(retion)", is a computer program which has been designed and developed by Roman Hovorka during his postdoctoral fellowship at the Centre for Measurement & Information in Medicine, City University, funded by Glaxo Research and Development, Ltd., and is available, at no charge, to interested investigators. A condition of this availability is that any manuscript and report based upon the ISEC software acknowledges the source of ISEC with a statement that ISEC can be obtained from the author, Roman Hovorka, PhD, Metabolic Modelling Group, Centre for Measurement and Information in Medicine, Department of Systems Science, City University, Northampton Square, London EC1V OHB, United Kingdom. DISCLAIMER THIS SOFTWARE IS PROVIDED ``AS IS'' AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL Roman Hovorka BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE. AIM OF ISEC ISEC estimates insulin secretion from plasma C-peptide concentrations. AIM OF MANUAL The manual shows how to use ISEC. An overview of methods adopted by ISEC is given. No special knowledge is required except for familiarity with the PC environment. NOTE Due to a demand for a revised version of the manual a working version has been produced

which does not include technical and implementation details. Such details will be reported separately or in an updated version of the manual.

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CONTENT 1. INSTALLING ISEC ............................................................................................................ 3 2. HARDWARE REQUIREMENTS ....................................................................................... 4 3. RUNNING ISEC ................................................................................................................. 3.1 First Input File ...................................................................................................... 3.2 Second Input File .................................................................................................. 3.3 Output File ............................................................................................................ 4. INSULIN SECRETION ...................................................................................................... 4.1 Introduction ........................................................................................................... 4.2 Co-secretion of Insulin and C-peptide .................................................................. 4.3 Plasma C-peptide as Indicator of Insulin Secretion ............................................ 4.4 Measurement Error .............................................................................................. 5. ISEC OVERVIEW ............................................................................................................... 5.1 Regularisation Method of Deconvolution Constrained to Non-Negative Values .............................................................................................................. 5.2 C-peptide Kinetics ................................................................................................. 5.3 Segmentation of Study Period .............................................................................. 5.4 Study Sub-Periods ................................................................................................. 5.5 Basal Secretion ...................................................................................................... 5 5 5 5 6 6 6 6 6 7 7 7 7 7 8

6. EXAMPLE RUN OF ISEC.................................................................................................. 9 6.1 First Input File TEST ......................................................................................... 10 6.2 Second Input File MODE.................................................................................... 11 6.3 Output File RESULTS ........................................................................................ 12 7. REMARKS ......................................................................................................................... 7.1 Assumptions ........................................................................................................ 7.2 Point-Area Deconvolution and ISEC .................................................................. 7.3 Execution Time.................................................................................................... 7.4 Implementation and Portability ......................................................................... 7.5 Missing Values .................................................................................................... 7.6 User's Estimation of the Measurement Error .................................................. 7.7 Format Notes ....................................................................................................... 14 14 14 14 14 14 14 15

APPENDIX A ........................................................................................................................ 16 A.1 Run-Time Errors................................................................................................. 16 ACKNOWLEDGMENT ........................................................................................................ 18 REFERENCES ...................................................................................................................... 19

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1. INSTALLING ISEC To install the program, copy ISEC.EXE from the distribution disk into an arbitrary directory on the hard disk of your computer.

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2. HARDWARE REQUIREMENTS The version of ISEC provided by the author requires an IBM PC compatible computer with MS-DOS version 4.0 or higher, an 80386 processor or higher, and at least 340 kB RAM. A numerical co-processor is not required although it is recommended as heavy numerical calculations are involved.

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3. RUNNING ISEC Choose the directory where ISEC is installed as the default directory, type ISEC followed by the names of two input files and the name of an output file. Once started, ISEC reads data from the two input files and reports its results in the output file. No interaction between the program and the user is required during the execution of the program. ISEC does not have a graphical output. 3.1 First Input File The first input file contains identification of the data set, the subject's sex, classification, age, height, weight and the time course of C-peptide concentrations. Classification indicates whether the subject is normal, obese (weight > 115% ideal body weight) or has non-insulin dependent diabetes mellitus (NIDDM). 3.2 Second Input File The second input file indicates the error of the C-peptide measurements and the segmentation of the study period. The error of C-peptide measurements is represented as a coefficient of variation (CV). 3.3 Output File The results of the calculations are reported in the output file. The file contains identification of the data set, error assessment, and the time course of insulin secretion and C-peptide concentrations.

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4. INSULIN SECRETION 4.1 Introduction Insulin is secreted by beta-cells in the pancreas. Direct measurement of the rate of insulin secretion requires catheterisation of the hepatic portal vein and is therefore not suitable for typical clinical conditions. Indirect estimation employing plasma insulin concentration fails due to large and variable hepatic extraction of insulin. In normal subjects, insulin secretion varies greatly during the day and is modulated by many plasma-born substances among which plasma glucose is the major determinant. Insulin secretion is impaired in patients with non-insulin dependent diabetes mellitus (NIDDM). Normalisation of insulin secretion is beneficial to patients with NIDDM. Quantification of insulin secretion allows an assessment of pancreatic responsiveness to be made. It is therefore possible to test the effect of various treatments on insulin secretion. Naturally, many other clinical/research situations exist in which the quantification of insulin secretion is useful. 4.2 Co-secretion of Insulin and C-peptide In 1969, Rubenstein et al (1) showed that insulin and a polypeptide known as C-peptide are secreted by beta-cells in a one-to-one molar ratio. Unlike insulin, C-peptide is not cleared by the liver (2) and its plasma concentration is a good indicator of secretion. In this manual, C-peptide secretion and insulin secretion are interchangeable as they are numerically equal in molar terms. 4.3 Plasma C-peptide as Indicator of Insulin Secretion Plasma C-peptide concentration is an indirect indicator of insulin secretion. As the kinetics of C-peptide are linear (3), secretion is proportional to plasma concentration during steady-state conditions. Under non-steady-state conditions, however, this simple relationship does not hold. In such a case, C-peptide plasma concentration is affected not only by 'immediate' secretion but also 'previous' secretion. Plasma concentration at any given time results from the accumulation of C-peptide in plasma due to immediate and past secretion. This accumulation process is usually denoted by the mathematical term 'convolution'. To reconstruct secretion from plasma concentration, an inverse operation has to be adopted. Not surprisingly, the operation is called de(-)convolution and ISEC adopts one representation of this inverse operation named the regularised method of deconvolution (4,5). 4.4 Measurement Error In the case of C-peptide, the measurement error has a great impact on the deconvolution of the secretion profile. When the measurement error is not taken into consideration, e.g. when point-area deconvolution method is employed, deconvolution under frequent sampling of plasma C-peptide (1 to 30 minutes sampling frequency) gives unrealistic oscillations in the time course of insulin secretion. The frequency of oscillations usually reflects the sampling frequency. Often, negative secretion values are calculated.

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5. ISEC OVERVIEW 5.1 Regularisation Method of Deconvolution Constrained to Non-Negative Values ISEC implements a regularisation method of deconvolution (4,5) constrained to nonnegative values. ISEC produces a non-negative secretion profile which satisfies the two following conditions. First, plasma concentration calculated from the secretion profile differs from the measured concentration by a pre-defined quantity. Second, the secretion profile has minimum oscillations, as quantified by a norm of second differences, among those profiles which satisfy the former condition. The first condition indicates that the secretion profile does not have to result in plasma concentrations which exactly match the measured concentrations. The difference is however pre-defined and usually represents the measurement error. In general, there are many (in fact an infinite number of) secretion profiles which satisfy the former condition. A preference favouring profiles with low oscillations is adopted by ISEC. This results in peaks and troughs in the time course of insulin secretion, caused by the measurement error, to be 'smoothed out'. Note that 'true' secretion peaks and troughs are not smoothed out under the condition that the measurement error is small when compared to the increase (decrease) in plasma concentration resulting from the change in secretion. Naturally, changes resulting in deviation of plasma concentration within the measured error cannot be identified. 5.2 C-peptide Kinetics Plasma C-peptide concentration results from two simultaneously occurring processes, Cpeptide appearance (secretion) and C-peptide disappearance (disposal). In principle, plasma concentration alone is not sufficient to estimate appearance without the knowledge of the disappearance and vice versa. ISEC determines the C-peptide disappearance model (kinetic model) from subject's individual data. The determination is based on a population-based study (6) which has shown that the parameters of the Cpeptide kinetic model can be approximated from the subject's weight, height, age, sex and classification (normal, obese, NIDDM). The use of approximated parameters does introduce an error though. However, it has been shown that this error is comparable to the variations due to a duplicate experimental assessment of the parameters (6). 5.3 Segmentation of Study Period ISEC requires the study period to be divided into distinct segments. Insulin secretion is calculated as a step-wise function given the segmentation. The segmentation does not have to follow the sampling schedule although it usually will have the form of the refined sampling schedule. A fine segmentation should be adopted when rapid changes in insulin secretion are expected, a crude segmentation can be adopted when insulin secretion is expected to change slowly. 5.4 Study Sub-Periods

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A study often includes landmark points which divide the study into two or more distinct sub-periods. Most studies include basal and post-intervention periods, for example. Smoothing of the secretion between the two sub-periods is often an unwanted feature as our a priori expectation is that there might be a sudden change in insulin secretion. ISEC utilises this a priori knowledge of the study design. Oscillations in C-peptide secretion are quantified separately for each study sub-period. In consequence, the calculated time course of C-peptide secretion loses its 'smoothness' at landmark points. No limit to the number of sub-periods is imposed by ISEC. 5.5 Basal Secretion Basal secretion is often of importance. It can be defined as a constant insulin secretion which results in basal C-peptide concentration. Smoothing sometimes interferes with the calculation of the basal secretion. To avoid this interference, ISEC includes a feature which allows the basal secretion to be calculated correctly without the interference of smoothing. To employ this feature, a 'basal' C-peptide measurement must be specified. Such a measurement is then treated by ISEC as a 'perfect' measurement with the measurement error approaching zero. The constrained deconvolution method is then forced to fit the basal C-peptide concentration and produces the correct basal secretion. Note that in the current version of ISEC only one C-peptide measurement can be specified as basal. Should several basal C-peptide concentration been measured experimentally, a mean value should be calculated and supplied to ISEC.

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6. EXAMPLE RUN OF ISEC Select the directory where ISEC is installed to be the default directory. Copy files TEST and MODE which are included on the distribution disk to this default directory (TEST contains the time course of C-peptide concentration measured in a normal subject following a meal at time zero). Type
ISEC TEST MODE RESULTS

Allow about 40 seconds execution time on a 486DX 33 MHz PC-AT compatible computer during which CODE prints on the screen various symbols indicating the progress of the calculations. Results will then be stored in the file RESULTS.

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6.1 First Input File TEST

ID: TEST-DATA SEX: M SUBJECT: normal AGE: 30 years HEIGHT: 179 cm WEIGHT: 81.1 kg TIME (min) C-PEPTIDE (pmol/l) 0 700 basal 15 800 30 1170 45 2010 60 1550 75 1510 90 2170 120 1240 140 1020 180 950 210 720

The first line indicates the name of the data set to be TEST-DATA. An arbitrary string with no blanks is accepted. The string should not be longer than 100 characters. The second line indicates the subject to be M(ale). To indicate the opposite sex, use F(emale). The third line indicates the patient's classification to be normal. On of the three categories must be specified: normal, obese or niddm, which stands for normal subjects, obese subjects (body weight > 115% ideal body weight), and patients with NIDDM, respectively. The next three lines indicate age, height and weight, respectively. The time course of C-peptide measurements follows. Each line contains the time of a measurement and the measured C-peptide concentration in pmol/l. Missing values are specified using a -999.0 value in the C-peptide measurement column. The basal keyword indicates that the measurement at time 0 should be considered 'perfect', i.e. with the measurement error approaching zero. basal can be specified only for the first measurement.

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6.2 Second MODE

Error-CV: Constraint: Reporting: Segmentation 0 break 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210

Input

File The first line indicates that the measurement error is 5%. The second line is included for historical reasons. The third line indicates the time course of C-peptide concentration calculated by ISEC should be reported every 5 minutes. The specification of segmentation follows. There are 43 segments defined: prior to 0 min, interval 0 to 5 min, interval 5 to 10 min, ... ... interval 205 to 210 min. A landmark point dividing the study into two subperiods is defined using the break keyword at time zero.

5.0 % non-negative 5 min

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6.3 Output File RESULTS

ISEC V3.4a June 1994 ID: TEST-DATA Date: Thu Oct 20 20:52:05 1994 CV wanted: 5 % CV achieved: 5.00 % Constraint: LINEAR Estimated insulin secretion: time (min) from to 0.0 0.0 5.0 5.0 10.0 10.0 15.0 15.0 20.0 20.0 25.0 25.0 30.0 30.0 35.0 35.0 40.0 40.0 45.0 45.0 50.0 50.0 55.0 55.0 60.0 60.0 65.0 65.0 70.0 70.0 75.0 75.0 80.0 80.0 85.0 85.0 90.0 90.0 95.0 95.0 100.0 100.0 105.0 105.0 110.0 110.0 115.0 115.0 120.0 120.0 125.0 125.0 130.0 130.0 135.0 135.0 140.0 140.0 145.0 145.0 150.0 150.0 155.0 155.0 160.0 160.0 165.0 165.0 170.0 170.0 175.0 175.0 180.0 180.0 185.0 185.0 190.0 190.0 195.0 195.0 200.0 200.0 205.0 205.0 210.0 secretion (pmol/kg/min) 2.16 1.92 2.58 3.26 4.03 5.06 6.36 7.70 8.43 8.16 6.93 5.49 4.45 4.16 4.60 5.64 6.96 7.92 8.09 7.32 6.01 4.50 3.08 1.99 1.39 1.30 1.50 1.84 2.18 2.43 2.61 2.72 2.77 2.77 2.72 2.63 2.51 2.36 2.20 2.01 1.82 1.61 1.41 break 90.0 95.0 2000.0 2027.0 2170.0 -170.0 7.8

The first line indicates the version of ISEC and its release date. The second line repeats the name of the data set. The third line specifies the date and time of the run. The fourth line repeats the CV specified in the second input file (5%). The fifth line reports the CV achieved by ISEC (also 5%). The two values should be equal. If different, the user has either underestimated or overestimated the error in the C-peptide measurements, or ISEC could not fit the measured C-peptide concentrations using the specified segmentation. The latter case is probably due to the segmentation being too 'crude'. As a rule of thumb, the segmentation should include at least one segment per Cpeptide measurement. The sixth line is included to support further development of ISEC. A list of insulin secretion rates follows. The first rate indicates secretion 2.16 pmol/kg/min prior to 0 min. of

Fit to C-peptide concentration: time (min) 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 calculated (pmol/l) 700.0 682.4 719.8 792.3 894.8 1036.9 1226.0 1451.0 1657.2 1782.7 1790.3 1710.0 1600.5 1522.3 1511.0 1581.2 1722.0 1882.3 measured (pmol/l) 700.0 800.0 1170.0 2010.0 1550.0 1510.0 diff. (pmol/l) -0.0 -7.7 56.0 -227.3 50.5 71.2 CV (%) 0.0 basal 1.0 4.8 11.3 3.3 4.7

The rate on the next line indicates secretion in the segment 0 to 5 min. Secretion of 1.92 pmol/kg/min was calculated in this segment. Rates on the following lines interpreted in a similar way. should be

The break keyword is included to confirm the input specification. The same applies for the keyword basal in the list below. A second list follows. It contains the time course of C-peptide concentration reported every five minutes. The report frequency is as

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100.0 105.0 110.0 115.0 120.0 125.0 130.0 135.0 140.0 145.0 150.0 155.0 160.0 165.0 170.0 175.0 180.0 185.0 190.0 195.0 200.0 205.0 210.0 1959.4 1816.9 1630.5 1435.5 1264.7 1140.6 1064.7 1027.1 1013.8 1011.2 1011.9 1012.0 1009.1 1002.0 990.0 972.6 950.0 922.2 889.5 852.4 811.4 767.1 720.1

specified in the second input file. The list can be regarded as the 'fit' to the measured Cpeptide concentration.

1240.0

24.7

2.0

1020.0

-6.2

0.6

950.0

0.0

0.0

720.0

0.1

0.0

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The list also contains measured concentrations, differences between measured and calculated concentrations, and the differences represented as the coefficient of variation (CV)

CV =

| measured - calculated | x 100 % measured

The average CV is indicated by the CV achieved heading. It is calculated according to the formula

CV achieved =

1 N

( measured i - calculated i )2 x100 % 2 measured i i =1

N

where N is the number of measurements. The 'perfect' basal measurement is not included in the calculation of CV achieved. The results, measured C-peptide concentrations, ISEC calculated concentrations, and estimated insulin secretion are summarised in Fig. 1. C-peptide

Figure 1. Calculated insulin secretion. Plot includes C-peptide measurements (n), insulin secretion (solid line) and model fit to C-peptide concentration (dotted line).

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7. REMARKS 7.1 Assumptions The following set of assumptions is adopted by ISEC in calculating insulin secretion: 1) C-peptide kinetics are described by a two-compartment model (7). 2) The parameters of the model of C-peptide kinetics are approximated from the subject's weight, height, age, sex and classification (6). 3) The measurement errors are uncorrelated, normal with zero mean, and with a constant coefficient of variation. 7.2 Point-Area Deconvolution and ISEC ISEC can be used to calculate insulin secretion using the point-area deconvolution method. To carry out the point-area deconvolution, specify a zero value for Error-CV in the second input file and indicate a segmentation equal to the sampling schedule. The calculated time course of insulin secretion then results in C-peptide plasma concentrations which closely match measured concentrations. 7.3 Execution Time The number of segments is the major determinant of the execution time. As a rule of thumb, the calculation time is linearly related to the number of segments, e.g. doubling the number of segments results in an two-fold increase in the execution time. In the example run, there are 43 segments and the calculations took about 40 seconds on a 486DX 33 MHz PC-AT compatible computer. Halving the segment length would result in a two-fold increase in the number of segments and a corresponding a two-fold increase in the execution time. 7.4 Implementation and Portability ISEC is written in C++ language for the Borland C++ compiler Version 3.1. No testing has been performed to investigate the portability to other C++ compilers. The source code is the property of the author and no modifications or permanent changes should be made without the written consent of the author. 7.5 Missing Values ISEC allows missing values to be specified. To indicate a missing value, use a -999.0 as the value in the C-peptide measurement column in the first input file. 7.6 User's Estimation of the Measurement Error The intra-assay error associated with C-peptide measurements, expressed as the coefficient of variation (CV), is at best around 4%. It is recommended to increase this error by at least 1% considering errors from other error sources, e.g. the error in sample dilution and unreported sampling time deviations. A value of at least 5% (depending on the C-peptide assay adopted) should be therefore used in the Error-CV data item in the

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second input file. 7.7 Format Notes The format of the two input files is described in the example. The following additional notes apply, namely: 1) Any number of spaces (" ") is allowed whenever a single space is acceptable. 2) Capital and lower case letters are interchangeable.

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APPENDIX A A.1 Run-Time Errors During execution, ISEC may terminate with an error message. A list of error messages (in italics) is listed below together with a short explanation (normal text) of the nature of the error. Use: ISEC <input_file_1 name> <input_file_name 2> <output_file name> ISEC was called with less than three parameters Cannot open input file <file_name> A file with the name specified cannot be opened for input. Check the existence of the file. Cannot open output file <file_name> A file with the name specified cannot be opened for output. Check the availability of space on your computer or existence of a file path, if this is used to specify the name of the output file. ID section missing The ID section of the first input file is missing/incorrect. Check the first input file. SEX section missing The SEX section of the first input file is missing/incorrect. Check the first input file. Bad SEX description A wrong identifier of sex. Use M(ale) or F(emale) in the first input file. SUBJECT section missing The SUBJECT section of the first input file missing/incorrect. Check the first input file. Bad SUBJECT description A wrong identifier in the SUBJECT section of the first input file. Use normal, obese, or niddm. AGE section missing The AGE section of the first input file is missing/incorrect. Check the first input file. HEIGHT section missing The HEIGHT section of the first input file is missing/incorrect. Check the first input file. WEIGHT section missing The WEIGHT section of the first input file is missing/incorrect. Check the first input file. TIME C-PEPTIDE section missing The label line TIME C-PEPTIDE is missing/incorrect in the first input file. Check the first input file. TIME C-PEPTIDE section: "BASAL" can be specified only for the first measurement Only the first measurement can be specified as 'basal'. Check the first input file.

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Nonnumeric values in TIME C-PEPTIDE section A non-numeric value occurred in the time course of C-peptide concentration. Check the first input file. ERROR section missing/incorrect The ERROR section of second input file is missing/incorrect. Check the second input file. CONSTRAINT section missing/incorrect The CONSTRAINT section of the second input file is missing/incorrect. Check the second input file. REPORTING section missing/incorrect The REPORTING section of the second input file is missing/incorrect. Check the second input file. SEGMENTATION section missing The SEGMENTATION section of the second input file is missing/incorrect. Check the second input file. Nonnumeric values in SEGMENTATION section A non-numeric value occurred in the SEGMENTATION section of the second input file. Check the second input file. No memory in heap No additional memory available on the program heap. Mostly due to too many segments specified in the second input file. As a rule of thumb, having 600 kB of continuous RAM available, it should be possible to specify up to 1000 segments. Root must be bracketed in RTSAFE The initialisation of non-linear regression analysis failed probably due to too few (or wrongly spaced) segments. Try to modify the segmentation in the second input file. Maximum number of iterations exceeded in RTSAFE Non-linear regression analysis could not converge with a sufficient precision. Try to modify segmentation (decrease the number of segments). ANY OTHER ERROR e.g. Bad dimensions in "+" Bad dimensions in "-" Bad dimensions in "*" etc. Internal errors which should not occur. If they do, try to modify segmentation. If it does not help please send the two input files which caused the run-time error to the author. An effort will be made to remove the potential bug from the next release.

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ACKNOWLEDGMENT Thanks to my colleagues Drs Malcolm A Young, Paul Soons, and Tom R Hennessy for their help during the preparation of this manual and for stimulating ideas which, I believe, have improved the performance and the user-acceptance of ISEC. Though it is hard for me to say I am nevertheless the only person responsible for all the errors and the omissions present in the user manual and in the program. Special thanks to Dr David JA Eckland for critical but constructive discussions which stimulated further developments of ideas adopted by ISEC. Thanks also to Prof Ewart R Carson and Dr Alan Bye for continuous support during my work on ISEC.

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REFERENCES 1. Rubenstein, A. H., J. L. Clark, F. Melani, and D. F. Steiner. 1969. Secretion of proinsulin C-peptide by pancreatic beta cells and its circulation in blood. Nature 224:697-699. 2. Polonsky, K. S., J. B. Jaspan, W. Pugh, D. Cohen, M. Schneider, T. Schwartz, A. R. Moossa, H. Tager, and A. H. Rubenstein. 1983. Metabolism of C-peptide in the dog: in vivo demonstration of the absence of hepatic extraction. J. Clin. Invest. 72:1114-1123. 3. Polonsky, K. S., B. H. Frank, W. Pugh, A. Addis, T. Karrison, P. Meier, H. Tager, and A. H. Rubenstein. 1986. The limitations to and valid use of C-peptide as a marker of the secretion of insulin. Diabetes 35:379-386. 4. Twomey, S. 1965. The application of numerical filtering to the solution of integral equations encountered in indirect sensing measurements. J. Franklin Inst. 279:95-109. 5. Press, W. H., S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery. 1992. Numerical Recipes in C. Cambridge University Press, Cambridge. 6. Van Cauter, E., F. Mestrez, J. Sturis, and K. S. Polonsky. 1992. Estimation of insulin secretion rates from C-peptide levels: Comparison of individual and standard kinetic parameters for C-peptide clearance. Diabetes 41:368-377. 7. Eaton, R. P., R. C. Allen, D. S. Schade, K. M. Erickson, and J. Standefer. 1980. Prehepatic insulin production in man: kinetic analysis using peripheral connecting peptide behaviour. J. Clin. Endocrinol. Metab. 51:520-528.