Physiological amino acid data management: Quantitation, assessment, reporting and storage

Comput. Biol. Med. Vol. 18, No. 6, pp. 431-439. Printed m Great Britain.

1988 0

OOIO-4825/88 53.00 + .tM 1988 Pergamon Press pk

PHYSIOLOGICAL AMINO ACID DATA MANAGEMENT: QUANTITATION, ASSESSMENT, REPORTING AND STORAGE ROBERTJ. CARTER,*TREVORLUKEY~and FLOYDF. SNYDER*$ * Biochemical Genetics Laboratory, Alberta Children’s Hospital, 1820 Richmond Road S.W., Calgary, Alberta, T2T 5C7; and Departments oft Computing Science and $ Pediatrics and Medical Biochemistry, University of Calgary, Calgary, Alberta, T2N 4N1, Canada (Received 14 October

1987;in revised form 25 March 1988; receiuedJor publication 14 April 1988)

Abstract-A series of routines, written in BASIC, have been developed to aid in the analysis, reporting and storage of physiological amino acid data used in the diagnosis and management of inherited metabolic disorders. The concentrations of 44 compounds are determined for three types of physiological samples: plasma, urine or cerebral spinal fluid. The programs facilitate the editing of numerical data, the creation ofa patient and sample information file to be merged with the results, the flagging of abnormal results, the addition of diagnostic or interpretive comments and the generation of hard copy reports. Files containing the foregoing information provide records which may be manipulated using data base programs for further analysis. Amino acids Reporting

BASIC Data base

Patient file

Identification of abnormals

INTRODUCTION There are more than 100 inherited disorders that are associated with abnormalities in the synthesis, degradation or transport of amino acids [ 1,2]. The identification and quantitation of amino acids in physiological fluids has become increasingly important for the early detection of metabolic disorders and for the monitoring of amino acid levels in children with treatable metabolic disease [l-4]. The concentration of approximately 44 physiological primary and secondary amines are commonly determined for either plasma, urine or cerebral spinal fluid. Amino acid analyzers are usually interfaced to an integrator or microcomputer in order to facilitate amino acid identification by retention time and quantitation by comparison to an analytical standard of known concentration. We have developed a series of routines, which complement our commercially obtained identification and integration package by facilitating the identification of abnormal amino acid profiles, the production of a final report and the creation of appropriate data base files. The commercially available programs [S] identify amino acids on the basis of their elution times, integrate the absorption signal from the analyzer and provide numerical output for each peak. The amino acid data is only of diagnostic value when linked to patient and specimen information, as well as interpretive comments. Prior to the development of the programs presented here, further clerical transcription and meshing of these components was required to generate a final report. The programs we have developed perform the following functions. (1) Editing of the analyzer integrated results. This includes correction of peaks misidentified on the basis of retention times, identifying co-eluting compounds possibly derived from medication and trace amounts of amino acids which are below the set threshold, and otherwise identifying peaks that cannot be quantitated for any reason. (2) Creation of a patient and laboratory information file and merging these with the appropriate amino acid results. (3) Examination of the results with a routine which compares patient amino acid concentrations to control data for either plasma, urine or cerebral spinal fluid and indicates on the monitor which values, if any, are outside the control ranges. (4) Addition of pertinent interpretative comments, diagnoses or further response to the physician CBM 18:6-D

431

ROBERT J. CARTERet al.

432

receiving the report. (5) Production of a final report including patient and laboratory information, quantitative results and comments. (6) Creation of files that contain all of the above information which can be read into any relational data base with the capability to import ASC II files. PROGRAM

DESCRIPTION

The following series of programs were designed by us to facilitate additional editing of the quantitative data generated by the Nelson programs, the entry and merging of patient and lab data, the addition of interpretive comments and the generation of a final report, as well as the production of files suitable for reading into many commercially available data bases. A flowchart illustrating the co-ordination of the various programs and their respective data files, including the Nelson and data base programs, is shown in Fig. 1.

D

[

Edlt.bas

1

x DBdeta.bss

Fig. 1. Composite diagram of the various programs indicating the respective files they generate and utilize.

Physiological amino acid data management

433

The Nelson system

The Nelson system [S] is a series of commercially obtained programs run on an IBM or compatible with a minimum of 192K bytes of memory. These programs (Fig. IA) can read analog data from two detectors and BCD data from an autosampler via an A/D converter cabled through a Ziatech IEEE-488 interface card and store the data. The data can be plotted, peak areas integrated, and sample concentrations calculated based on either internal or external standards. The operator controls how the program collects the data, identifies and integrates the peaks, calculates concentrations and reports the results, through a series of menu driven forms. Installation

of amino acid standard ranges for plasma, urine and cerebral

spinaljluid

The program Instalaa.BAS is menu driven (Table 1) and creates three separate data files (Plasma.DAT, Urine.DAT, and CSF.DAT) which are accessed by other programs (Fig. 1B). Contained in the data files are the maximum and minimum “normal” values for the different physiological samples, plasma, urine and cerebral spinal fluid. Normal amino acid ranges were initially obtained from those previously published for plasma [6-g], urine [lo-121 and cerebral spinal fluid [ 13- 151, and supplemented with our own findings. Access to entering, editing or printing the values in the different data files is done through this program, however, EDLIN (a component of IBM DOS) may be used so long as the format of the file is maintained.

Table 1. Menus A. Yenu

for

Instalaa.BAS

program

*~~kt*~~**~~X*~****X**V*t*t*Q*****~******* h

*

Y

*

* *

fc * * *

1) Enter plasma control ranges 2) Enter urinarv control ranges * 3) Enter CSF control ranges 9< 4) Edit a control range file sr 5) Print a control range file * 6) Exit from program 9< *t*****t****************************

8.

Menu common to

all

programs

AMINO

Enter

* * *

ACID

1.

File

Patient

2.

Edit

amino

3.

Print

4.

Exit

MENU Data acid

amino to

PROGRAM

acid

results results

System

number or use then press

arrows to move to

selection;

ROBERT J. CARTERet al.

434

Table 2. Patient and specimen information

1

PATIENT Name (25

2

PATIENT ID (6

3

DATE OF BIRTH (YY/MM/DD)

[87/01/071

4

Sti

[Ml

5

HOSPITAL ID (12

6

PHYSICIAN (25

7

LAB. ID (7

8

DATE RECEIVED (W~/DD)

C87/01/021

9

DATE COLLECTED (YY/MM/DD)

[87/01/01]

CHAR)

I PATIENT, Name

digits)

[ 1035461

(M/F) CHAR)

[ACH 198719871

CHAR)

[DOCTOR’S Name [87-19871

CHAR)

10 SPECIMEN (25

[PLASMA (FASTING)

CHAR)

11 REASON

FOR REFERRAL (60

E(DIT):

S(AVE)

;

N(EXT);

CHAR)

Q(QUIT)

[SEIZURES

I

:

All characters in bold print appear on the video terminal as reverse video

Entry of patient and sample information

The program DBdata.BAS creates (Fig. 1D) four data files by using a name supplied by the operator and appending a different letter to the end of the name for each file (Name.DAT, NameP.DAT, NameL.DAT and NameIDAT). The length of the file name is limited to seven characters. We have found it most convenient to use the month and day (i.e. FEB08). The files accept input by filling in a form generated on the screen by the program (Table 2). Before the form appears, the chromatogram number to which the information pertains must be entered. As each field in the form is filled in, the program provides a visual field length by reversing the video. Before allowing the operator to proceed to the next field, error checks are made for length of field, data type, and, in date fields, that the date was entered in the order of Year/Month/Day. If an error is present, the type of error is printed on line 23 and the cursor is returned to the beginning of the field deleting what was in the field. After all eleven fields are filled, the operator has the option to edit any field on the screen or to save the form. When each form is saved, the operator has the option to quit. By quitting, the data files are closed and no more information can be appended to the files. Edlin may be used to correct any typographical errors that the operator missed so long as the format of the files and length of fields are maintained. As each form is filled, it is appended to the end of the files. The first data file (Name.DAT) stores the information from the form as it appears on the screen and is used later by another program to allow the operator to make comments on the amino acid profile and to generate a report. The second file (NameP.DAT) extracts only the patient information (i.e. patient name, sex, date of birth, etc) from each form as it is filled out and is only used for reading into a data base at a future time. The third file (NameL.DAT) is also for data base use and only extracts the lab information and the age of the patient (which is calculated by the program) at the time the sample was collected. In order to insure that the correct amino acid profiles are reported with the correct patient and sample information, a fourth file (NameLDAT) is created and contains the chromatogram number, patient number and lab sample number from each form as it was filled in. This file is used by the comment and report program.

Physiological

amino acid data management

435

Editing of integrated data acquired by the Nelson system The program Edit.BAS (Fig. 1C) is used to edit the amino acid results generated by the Nelson system and provides a method for reporting trace and co-eluting peaks. The following files must be present for the program to run: Plasma.DAT, Urine.DAT and CSF.DAT, which are generated by Instalaa.BAS and Achromatogram#.ATB and Bchromatogram#.ATB which are generated by the Nelson system. After entering the number of the chromatogram to be edited, the program retrieves the amino acid concentrations from the appropriate Achromatogram#.ATB file for the 570 nm wavelength results and the Bchromatogram#.ATB file for the 440 nm wavelength results and converts them to three significant figures. Once the operator enters the type of sample, the program compares the amino acids with the appropriate control range file and generates a form on the screen with each amino acid, and its concentration (Table 3). If assay values are outside the “normal” range a 7 or 1 appears beside the value. At this point, the operator can enter “---” for amino acids that are not detectable, “***” for amino acids that are co-eluting with an unknown peak, “N.D.” for an amino acid which is present but whose value cannot be correctly determined due to perturbations introduced by the analyzer itself, “TRACE” for amino acids that are present, but at concentrations below the set threshold limit or a new value calculated by the operator to correct for values being calculated by the Nelson system when the retention time for the amino acid is outside its proper window. After each edit, the program checks the amino acid concentration against the standard range data and changes the t or 1 accordingly. Once the operator is satisfied that the amino acid values are now correct, the results are saved to an Echromatogram#.DAT file for later processing by Rprter.BAS. Flagging of abnormal results, entry of diagnostic assessment and production of report

The program Rprter.BAS (Fig. 1E) has two parts; the first part for adding comments to reports and the second for printing hard-copies of the reports. The following files must be present for the program to run: Plasma.DAT, Urine.DAT and CSF.DAT which are generated by Instalaa.BAS; Name.DAT and NameI.DAT which are generated by DBdata.BAS; and Echromotogram#.DAT which is generated by Edit.BAS. After entering the name of the host file (name used in DBdata.BAS to create Name.DAT) and the name of the person who conducted the assay, the program creates NameC.DAT to sequentially hold the patient file number, the chromatogram number, lab sample number and the amino acid

Table 3. Amino acid data DATA FOR CHROMATOGRAM i/O843 PLASMA SAMPLE

1 2 3 4 5 6 7 8 9

PSF.R --TAUR-0.152 PJw4 0.021 UREA 2.091 ASP 0.028 HPRO TRACE THRE 0.035 SER 0.115 ASPN 0.072

ACCEPTABLE

10 11 12 13 lb 15 16 17 18

GLU GLUM SAFE AADA PRO GLY ALA CITR AABA

0.073 1.431f N.D. --0.200 0.196 0.097 3.201 f N.D.

19 20 21 22 23 24 25 26 27

VAL 0.097 CYS 0.028 MET.H 0.111 + CYST __ILEU 0.011 LEU *** NLEU .__TYR 0.034 BALA me-

28 29 30 31 32 33 34 35 36

PHEN BABA HCYS GABA ETH NH3 AHLY ORN LYS

O.U’:L 0.670 ----N.D. O.“,lr, --U.UW+ 0.136

37 38 39 40 41 42 43 44 45

IHEH HIS TRYP 3MEH ANSR CARN ARG

O.UW 0.116 0.001 -------TRACE )

EDITING

*** ND TR OK AN ACCEPTABLE NUMBER t ie iHi. rf ##f 1 ENTER THE NUMBER OF THE AMINO ACID TO BE EDITED FOLLOWED BY A COMMA AND THE CORKECTION (0,Q TO END EDITING)

All characters

in bold print appear

on the video terminal

as reverse video.

436

ROBERTJ. CARTER et al. Table 4. Patient

and amino acid data and interpretive

comments

PID 103546 PATIENT PATIENT, Name DOB 87/01/01 SEX M HOSPNUN ACH 19871987 PHYSICIAN DOCTORS Name LID 87-1987 RECEIVED 87/01/02 COLLECTED 87/01/01 SPECIMEN PLASMA (FASTING) REASON FOR REFERRAL SEIZURES

1 PSER ---

2 3 4 5 6 7 8

TAUR PEAN UREA ASP HPRO THRE SER 9 ASPN

0.152 0.021 2.09 0.028 TRACE 0.035 0.115

0.072

10 GLU 0.073 11 GLUM 1.43 f 12 SARC N.D.

19 VAL 20 CYS

13 MDA

---

14 15 16 17 18

0.200 0.196 0.097 3.20 t N.D.

22 23 24 25 26 27

PRO GLY ALA CITR AABA

21 MJZTH

CYST ILEU LEU NLEU TYR BALA

0.097 o.oa3 O.lllf

--_ 0.011

*** ___ 0.034 ___

28 PHEN 0.032 29 BABA 0.670

30 HCYS --31 GABA --32 ETH N.D. 33 NH3 0.314 34 AHLY --35 ORN 0.009) 36 LYS 0.136

37 1MEH 0.009

38 39 40 41 42 43 44 45

HIS TRYP 3MEH ANSR CARN ARG

0.116 0.001 ------'TRACE+

A(ccept); E(dit)? 1 Marked increase in plasma citrulline (approximately 80-fold), with 2 unknown peak in leucine area. Glutanine is 2-fold increased and 3 arginine is notably absent with low ornithine level. 4 Pattern is consistent with a diagnosis of citrullinemia and a 5 deficiency of argininosuccinate synthetase activity. 6) 7) 8) Allcharactersin boldprint appearon thevideoterminal asreverse video.

results by merging NameI.DAT and the appropriate Echromatogram#.DAT files in an order dictated by NameLDAT. The NameC.DAT file is now ready for use with Name.DAT for reporting and for using in a data base. At this juncture, the program creates NameR.DAT before retrieving the first set of patient data and amino acid data from Name.DAT and NameCDAT respectively, determines the sample type, compares it with the “normal” control values and displays all the data on the screen (Table 4). If any of the amino acid values are outside the “normal” range, arrows t or 1 will appear next to the result. The operator is now in the add comments mode and can provide text up to a maximum of 8 lines of 70 characters each. A return on an empty line or on the 8th line, places the operator into an accept/edit mode. If the comments are correct, they are saved to NameR.DAT and the operator has the option of quitting or viewing the next set of data in the file. If the program is ended, the NameR.DAT file is saved and upon re-entry into the program, the operator may edit comments in the NameR.DAT file, add additional comments to the file or use the file along with Name.DAT and NameC.DAT to produce a hard-copy report. In the report mode, the operator can choose to automatically print 4 copies of each report (Table 5) in the file or to manually enter the lab number of a particular report and the number of copies to be printed. In the manual mode the operator is continually given the choice to report another record or to quit. DATA

BASE

PROCESSING

Any relational data base that can import ASCII files and interrelate three data base files, may be used (Fig. 1F). The three data files are NameP.DAT, NameL.DAT and NameC.DAT and contain the primary key field as the patient file number. A secondary key field in NameL.DAT and NameC.DAT contains the chromatogram number of that particular lab data. Because the three files are ASCII files, we are able to transfer them across telephone communications and use the MRDS data base program on a Honeywell mainframe. By this means we have been able to obtain age and sex matched control ranges for different amino acids when studying dietary restricted [3] or supplemented [4] patients. We have also imported the files into the Paradox data base [16] without further modifications and now store results in house.

Physiologicalamino acid data management

431

Table 5. Report of amino acid analysis PID :103546 CHROM: LAB : DATE COLLECTED: DATE RECEIVED: DATE REPORTED:

PATIENT, Name M SEX : BIRTH DATE : 87/01/01 HOSP. : ACH 19871987 SPECIMEN: PLASMA(FASTING) REFERREDBY : DOCTOR’SName REASONFOR REFERRAL: SEIZURES Name :

AMINOACIDS _____--____

mM

Phosphoserine .............. Taurine .................... Phosphoethanolamine ........ Urea ....................... Aspartic Acid .............. Hydroxyproline ............. Threonine .................. Serine ..................... Asparagine ................. Glutamic Acid .............. Glutamine .................. Sarcosine .................. alpha-Aminoadipic Acid ..... Proline .................... Glycine .................... Alanine .................... Citrulline ................. ..... alpha-Amino-n-Butyrate Valine ..................... Cystine .................... Methionine ................. Cystathionine .............. *** - CO-ELUTING, COMMENTS :

mM

--0.152 0.021 2.09 0.028 TRACE 0.035 0.115 0.072 0.073 1.43 N.D. --0.200 0.196 0.097 3.20

Isoleucine ................. Leucine .................... Norleucine ................. Tyrosine ................... beta-Alanine ............... Phenylalanine .............. ...... beta-Aminoisobutyrate Homocystine ................ gamma-Aminobutvrate ........ Ethanolamine ............... Ammonia.................... ......... Allo-hydroxylysine Ornithine .................. Lysine ..................... .......... I-Methylhistidine Histidine .................. .......... 3-Methylhistidine ‘Trvptophan ................. Anserine ................... Carnosine .................. Arginine ...................

N.D.

0.097 0.028 0.111 ---

NINHYDRIN +ve

8U-fold), increased

arginine is notably absent wiLh low ornithine level. Pattern is consistent with a diagnosis of ciLrullinernia deficiency of argininosuccinatt! svnthetaw wtlvlt;.

copv

1 of

0.011 *** --0.034 --0.032 0.670 ----N.D. 0.314 --0.009 0.136 0.009 0.116 0.001 ------TRACE

PEAK

Marked increase in plasma citrulline (approximatelv unknown peak in leucine area. Glutamine is 2-told

ASSAYED BY

870843 87-1987 87/01/02 87/01/02 87/09/24

: TECH. Name

with and

and a

.._ ‘:s:tin_atSrw’L._.__ lnterpl eted bb

_

1

HARDWARE

AND

SOFTWARE

The programs we have written can run on an IBM-PC/XT/AT BASICA, version 3.0 and 192K of buffer are available.

or compatible so long as

SUMMARY The routines described here have now been in operation for three years and are used in the handling of approximately 1000 samples per year. These procedures represent a significant advantage over non-computerized records and reports for several reasons. Patient and specimen information may be entered at a secretarial workstation, quantitative results may be edited by laboratory technologists and the diagnostic evaluation or interpretation completed in an area with access to patient files and other source materials. The major objectives of these routines are the meshing of patient-specimen information, amino acid

ROBERTJ.CARTERetal.

438

results and diagnostic assessment, the generation of reports and storage in a retrievable format, The inspection of results is greatly facilitated by the internal comparison against standard ranges for the 44 compounds quantitated in each of the different types of physiological specimen. Thus the automated flagging of abnormal results alone represents a significant advantage in the quality and ease of assessment for such a complex array of results. The direct generation of reports without further transcription is also efficient and minimizes opportunities for introduction of errors. The retention of both patient information and amino acid results in a data base compatible format has already facilitated the retrieval, analysis and manipulation of data from 60 patients [3, 43 which otherwise would require visual inspection of amino acid results and patient files, manual retrieval and re-entry for further analysis. The procedures described here should also have broader applicability to any analytical system in which a spectrum of compounds are routinely analyzed such as in studies ofphysiological organic acids where profiles of more than 100 compounds are being assessed c171. Acknowledgements-This work was supported by a capital equipment grant from the Alberta Heritage Foundation for Medical Research, the Alberta Hereditary Disease Program, and the Medical Research Council of Canada. We thank Terry Harris for assistance in the preparation of this manuscript.

REFERENCES 1. D. Welner and A. Meister, A survey of inborn errors of amino acid metabolism and transport in man, Ann. Rev. Biochem. 50,911-968 (1981). 2. W. L. Nyhan, Abnormalities in Amino Acid Metabolism in Clinical Medicine. Appleton-Century-Crofts, Norwalk, Connecticut (1984). 3. H. Parsons, E. Fung and F. Snyder, Branched-chain alpha-keto acids for the diagnosis of maple-syrup-urine disease, New Engl. J. Med. 316,951 (1987). 4. H. G. Parsons, R. B. Scott, A. Pinto, R. J. Carter and F. F. Snyder, Argininosuccinic aciduria: long-term treatment with a&in+ J. Inher. Metab. Dis. 10, 152-161 (1987). 5. 3ooO Series Chromatography Data System, Version 3.6. Nelson Analytical, 10061 Bubb Road, Cupertino, California (1986). 6. J. C. Dickinson, M. A. H. Rosenblum and P. B. Hamilton, Ion exchange chromatography of the free amino acids in the plasma of the newborn infants, Pediatrics, Springfield 36, 2-13 (1965). 7. S. E. Snyderman, L. E. Holt, Jr, P. M. Norton, E. Roitman and S. V. Phansalkar, The plasma aminogram. I. Influence of the level of protein intake and a comparison of whole protein and amino acid diet, Pediat. Res. 2, 131-144 (1968). 8. H. Ghadimi and P. Pecora, Plasma amino acids after birth, Pediatrics, Springfield 34, 182-191 (1964). 9. M. D. Armstrong and V. Stave, A study of plasma free amino acid levels. II. Normal values for children and adults, Metabolism 22, 561-569 (1977). 10. D. K. Rassin. G. E. Gaul]. K. Heinonen and N. C. R. Raiha. Milk protein auantitv and quality in low birth weight infants. II. Effects’on selected aliphatic amino acids.in plaima and-urine,-Pediariics, Spring/ield 59, 407-422 (1977).

11. M. J. Carver and R. Paska, Ion exchange chromatography of urinary amino acids. I. Normal children, Clinica chim. Acta 6.721-724

f1961).

12. A. Scott-Emkakpor, J. .V.H&ins and A. F. Kohrman, Citrullinemia: a new case, with implications concerning adaptation to defective urea synthesis, Pediat. Res. 6, 626-633 (1972). 13. J. C. Dickinson and P. B. Hamilton, The free amino acids of human spinal fluid determined by ion exchange chromatography, J. Neurochem. 13, 1179- 1197 (1966). 14. T. L. Perry, S. Hansen, S. Diamond and D. Stedman, Plasma amino acid levels in Huntington’s chorea, Lancet 1,806-808 (1969). 15. M. van Sande, Y. Mardens, K. Adriaenssens and A. Lowenthal, The free amino acids in human cerebrospinal fluid, J. Neurochem. 17, 125-135 (1970). 16. Paradox, Version 2.0. Ansa Software, 1301 Shoreway Road, Belmont, California 94002, U.S.A. (1987). 17. R. A. Chalmers and A. M. Lawson, Organic Acids in Man. Analytical Chemistry, Biochemistry and Diagnosis of the Organic Acidurias. University Press, Cambridge (1982).

About the Author -ROBERT J. CARTERreceived a B.Sc. degree in Biochemistry from the University of Guelph in 1970 and the MSc. in Clinical Biochemistry from the University of Calgary in 1977. He is presently a Genetics Research Assistant, Biochemical Genetics Laboratory, Alberta Children’s Hospital, Calgary. About the Author -TREVOR LUKEYreceived a BSc. in Medical Biochemistry from the University of Birmingham in 1969, the MSc. in Biochemistry from the University of Birmingham in 1974 and a

Physiological amino acid data management M.Sc. in Computing Science from the University of Calgary in 1985. He is presently an Instructor in the Department of Computing Science at the University of Calgary. About the Author- FLOYD F. SNYDERreceived a BSc. degree from the University of Calgary in 1966, M.Sc. in organic chemistry from McMaster University in 1969 and Ph.D. in biochemistry from the University of Alberta in 1973. He is presently an associate professor in the Departments of Pediatrics and Medical Biochemistry, University of Calgary, and director of the Biochemical Genetics laboratory, Alberta Chiidren’s Hospital, Calgary.

439