CA1327993C - Nmr lipoprotein analysis of blood - Google Patents

Abstract

Abstract of the Disclosure A method and apparatus for analyzing blood plasma to determine the concentration of its lipoprotein constituents, VLDL, LDL, HDL and proteins includes obtaining the NMR chemical shift spectrum of a sample. Stored reference NMR spectra of the lipoprotein constituents are added together to form a lineshape that best fits the measured blood plasma NMR
spectrum, and from this, the concentration of each lipoprotein constituent in the blood plasma is determined.

Description

- --' 13 2 7 9 9 3 - ~R 88 P 7463 E~ AUS1.

~ NMR LIPOpROTEIN ANALYSIS OF BLOOD
. -~ck~rQun~ of the Inve~tlon The fleld of the invention is the measurement of lipoprotein level~ ln blood pla~ma or blood serum and, more particularly, the levels of low-density lipoprotein~ ~L~L), high-denslty llpoprotelns (HDL) and very low-density lipoproteins(VLDL). These lipoproteins account for the vast majorlty of the cholesterol found ln blood.
The importance of accurately meaauring cholesterol levels in blood is well known. ~he federal government, in combination with more than twenty health organizations, has launched an aggressive campaign, through the National Cholesterol Education erogram~ to convince phy~iclans and the general population of the dangera of high choleaterol levels in the blood. All person3 are urged to have thèlr cholesterol levels checked, and specific treatments are recommended based on the precise measured cholesterol level. In addltion, treatments are not based solely on the total chole~terol level, but lnstead, on the level of LDL cholesterol. LDL choleaterol appeara to be the ma~or cause of clogged arterles, whereas HDL cholesterol alds in removlng cholesterol deposits. A separate, and more expensive test ls requlred to determine the level of LDL
cholesterol and lt 19 usually not conducted unleas the measured total chole~terol level ls at the borderllne or hlgh rls~
levels.
Current methoda for measurlng choleaterol levels are notorlously lnaccurate and the standard practlce la to repeat the measurement a number of tlmeY when hlgh levels are detected on the flrst measurement. Inaccuracles o~ 5~ or more have been found ln nearly half of the measurements made by testlng laboratorles and 15~ of the measurements were lnaccurate by an amount greater than lO~. The~e inaccuracies are lnherent ln ', , , '~ ' :, ' '~ ' ~ . ' . . , . : . . ., , :

~- `' 1327993 the current measurement methods whlch require considerable handling of the blood and certain presumptions a~out the ratlos of its constituent parts.
Dlrect quantlzatlon of llpoproteln cholesterol ls usually achleved by enzymatic assay of the indlvidual lipoproteins, which are separated by ultracentrifugation, electrophoresis, or selective precipitation. There is great variability among the available separation methods in terms of accuracy, convenience, and cost. Generally, the most accurate methods are those involving ultracentrifugation, but these are very time consumlng and expensive and therefore not suitable for large-scale population studies. The most widely used alternative is an lndlrect method introduced by W.T. Friedewald, R.I. Levy, and D.S. Fredrickson, ln thelr publication "Estimation of the Concentratlon of Low-Denslty Llpoproteln Cholesterol ln Plasma, Without Use of the ~reparative Ultracentrifuge", Clin. Chèm., 1~, 499-502 ~1972). In thls procedure, plasma trlglycerlde ~TG) and total cholesterol ~TC) are mea~ured by en~ymatic assay. To a separate aliquot of plasma ls added one of several reagents which selectively preclpitates VLDL and LDL. After removing the precipitate by centrifugation, the supernatant is assayed for cholesterol to provlde a measure of HDL cholesterol ~DL-C). An estimate of VLDL cholesterol ~VLDL-C) ls then made by divldlng the plasma triglycerlde level by five. The LDL
cholesterol ~LDL-C) concentratlon ls then calculated by dlfference: LDL-C ~ TC - ~HDL-C + VLDL-C). Although thls method ls relatlvely rapld and lnexpensive, there are several steps where experlmental error can be lntroduced, particularly ln the preclpitation ~tep. In addltlon, the accuracy of the analysis depends on the aSsumptlon that VLDL-C can be rellably estlmated as one flfth the concent~atlon of plasma trlglycerlde. When fasting samples are used, this ls generally .

.

1327~33 ~0~65-2981 true~ but other formulas have also been suggested to give more accurate values as described by D.M. DeLong, E.R. DeLong, P.D.
Wood, K. Lippel, and B.M. Rifkind, in their publication "A
Comparlson of Methods for the Estimation of Plasma Low- and Very Low-Density Lipoprotein Cholesterol", J. Am. Med. Assoc., 256, 2372-2377 (1986).
Summary of the Invent1on The present invention relates to a method for measuring the lipoprote~n constituents of blood using a nuclear magnetic resonance (NMR) technique. Thus, in one aspect the present invention provides a method for measuring the lipoprotein constituents of blood, the steps comprising: storing the NMR
spectrum of each lipoprotein constituent as a reference spectrum for that constituent; acquiring the NMR spectrum of a plasma or serum sample of the blood to be analyzed; producing a calculated lineshape by adding together the reference spectrum for each constituent in amounts determined by respective constituent coefficients; adjusting the constituent coefficients to fit the calculated lineshape to the NMR spectrum of the sample; and 2~ calculatlng the concentration of at least one lipoprotein constituent as a function of the value of its constituent coefficient.
In another aspect, the invention provides apparatus for measuring a plurality of lipoprotein constituents of blood comprising: means for storing the NMR spectrum of each one of said plurality of lipoprotein constituents as a reference spectrum for that constituent; means for acquiring the NMR spectrum of a plasma or serum sample of the blood to be analyzed; means for producing a calculated lineshape by adding together the reference spectrum for each constituent in amounts determined by respective constituent coefficients; means for adiusting the constituent coefficients to fit the calculated llneshape to the NMR spectrum of the sample; and means for calculating the concentration of at least one of said plurality of lipoprotein constituents as a functlon of the value of its constituent coefficient.

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279~3 More specifically, the method includes acquiring proton NMR
data from a sample of blood plasma or serum, processing the acquired NMR data to produce a chemical shift spectrum, and deconvoluting the spectrum in terms of four standard lipoprotein constituent spectra to give the concentration of each of the four lipoprotein constituents. It has been discovered that the spectrum is accurately represented by a linear combination of the spectra of four constituents into which the blood can be fractionated. These .our constituents are VLDL, LDL, HDL and protein and their NMR spectral properties have been found to be virtually invariant from person to person. Thus, any differences in the NMR spectra are due entirely to differences in the amplitudes of the constituent spectra, which, in turn, is due to the concentrations of those constituents in the blood.
A general object of the invention is to provide an accurate and reliable measurement of the lipoprotein constituents of blood.
Since the observed spectrum of a whole plasma sample is closely ' simulated by appropriately weighted sums of the NMR spectra of its four lipoprotein constituents, it is possible to extract the concentrations of these constituents in a sample by calculating the weightlng factors 3a ; . . . .
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- which give the best fit between the sample spectrum and the calculated spectrum. The handling and processing of the sample is relatively simple compared to prior methods and there is, therefore, less opportunity for error. The sample ls merely prepared for the NMR measurement and the measurement is taken at a controlled temperature and at a controlled magnetic field strength.
Another object of the invention is to provide a method for measuring the lipoprotein constituents of blood at an ' economical cost and on a mass basis. The preparation of the sample is a triviaI task and the actual NMR measurement is carried out automatically by an NMR spectrometer in seven minutes or less. The deconvolution calculatlons are also carrie~ out automatically by a computer which prints out a report that indicates the concentrations of the lipoprotein constltuents.
Yet another ob~ect of the invention i9 to improve~ the accuracy of the deconvolution''p'roc,e~s by accountlng for non-llpoprotein constituents. Standard NMR re~erence spectra for metabolltes such as lactate, vallne and hydroxybutyrate are produced and these are used along wlth the NMR re~erence spectra for the lipoprotein constituents to deconvolute the sample UMR spectrum.
The foregolng and other ob~ect~ and ad~antages of the inventlon wlll appear from the followlng descriptlon. In the description, reference i9 made to the accompanylng draw~ngs whlch form a part hereof, and ln whlch there ls shown by way of lllustratlon a pre~erred embodlment Or the lnvent~on. Such embodlment does not neces~arlly repre~ent the rull ~cope of the lnventlon, however, and rererence 18 made therefore to the clalms hereln for interpretlng thé scope of the lnventlon.

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-` 1327~93 , rl ef De~cr1~tion of the Drawln~s Flg. 1 is a graph showing the chemical shift spectra of a first plasma sample and lts lipoprotein constituents;
Flg. 2 is a graph showing the chemical shift spectra of a different plasma sample and lts correspondlng lipoproteln constituents:
Fig. 3 is a graph showing the chemical shift spectra of a third plasma sample and its corresponding lipoprotein and non-lipoprotein constituents; and ~0 Fig. 4 is a block dlagram of the apparatus employed to practice the present invention.

General Descr;ptton of the T~n~i~n lH NMR spectra of human blood plasma contain two prominent peaks centered at approximately 1.2 and 0.8 ppm ~relative to the chemical shift standard, TSP~, These peaks ar~se from methylene (CH2) and methyl (CH3) protons, respectively, of plasma lipids. Each of these peaks 19 very heterogeneous ln nature, consLstlng of overlapplng resonances from protons of the several chemically dlstlnct classes o~ liplda present ln plasma: trlgylcerldes; cholesteroli cholesterol esters; and phospholipids. These liplds are packaged together into three ma~or types o~ llpoproteln particles, which differ ln the proportLon4 of llpld4 whlch they contaln. These llpoprotein partlcles also differ ln denslty from whlch thelr names are derlved: very low dens~ty llpoproteln (VLDL), low density llpoprotein (LDL), and hlgh denslty llpoproteln ~HDL). Only that ~raction of the llpids ln these llpoprotein partlcles that are in a ~luld, moblle state (as opposed to an ordered llquid-crystalllne atate) contrlbute NMR plasma resonances. The heterogenelty o~ these slqnals i9 re~lected by thelr complex llneshapes, whlch vary ~rom peraon to person owlng to .
:
.

-1 ~27993 - varlatlons of the plasma concentratlons of the dlfferent lipoproteln particles, each of which has its own characterlstlcally dlfferent NMR spectral propertles.
The method of the present invention allows the , 5 concentratlons of all three llpoprotein particles (VIDL, LDL, HDL) of a plasma sample to be extracted from its lH NMR
spectrum by a computer analysls of the lineshapes of lts methyl and methylene ~ignals. The method exploits the finding that thls region of the observed plasma spectrum is accurately-represented by a simple linear combinatlon of the spectra of four constituents into which plasma can be fractionated by dlfferentlal flotatlon ultracentrlfugatlon. The four conatituents are differentiated on the basis of their density and lnclude: VLDL ~density < 1.006); LDL ~den~lty = 1.006 to 1.063); HDL (density - 1.063 to 1.21); and "Protein" (density >
1.21). The latter constltuent 19 the moatly protein-containlng bottom fraction left behlnd after flotatlon of the lipoproteins.
The NMR spectral propert~es of these constltuent3 have been found to be vlrtually lnvarlant ~rom person to person.
Thls ls illustrated in Table 1 which ls the result of a study - conducted at the Vnlverslty of Wlsconaln-M~lwaukee and the ~ Medlcal College of Wisconsln.

TA1~(T~

500 MHz NM~ Parameters of the Separated Llpoproteln Constituents of Plasma ~9~ M~an + Sl~
j YL~L (n-117) ~ CH2 Chemlcal Shlft (ppm)1.233 ~ 0.002 i CH3 Chomlcal Shlft ~ppm)0.839 ~ 0.002 ~ 35CH2 Llnewldth ~Hz) 20.3 + 1.9 ; CH3 Llnewldth (Hz) 16.3 ~ 0.8 ~ , CH2/CH3 Intenalty ~atlo 3.76 + 0.29 -.: :

:~ ,. . . . -,.~, . .

T.nr. (n=66) CH2 ChemLcal Shift (ppm) 1.219 + 0.005 CH3 Chemlcal Shift (ppm) 0.822 + 0.002 ; 5 CH2 ~lnewldth IHz) 34.0 ~ 2.9 CH3 Linewidth (Hz) 21.1 + 1.0 CH2/CH3 In~ensity Ratlo 1.27 i 0.13 (n~70 CH2 Chemlcal Shlft (ppm) 1.186 i 0.004 CH3 Chemlcal Shift ~ppm) 0.796 ~ 0.003 CH2 Linewidth ~Hz) 34.4 i 2.9 CH3 Llnewidth (Hz) 20.0 + 0.8 15 CH2/CH3 rntensity Ratio 1.58 i 0.13 ~BQ5EI~ (n=lll) CH2/CH3 Intenslty Ratio 0.37 + 0.10 Thus, dlfferences among the NMR signals from the plasma of individuals are caused by differences in the~ amplitudes of the lipid resonances from the four constltuents which ln turn are proportional to their concentrations in the plasma.
This ls lllustrated in Flga, 1 and 2 ln which the NMR
chemical shift spectra of two sùbstantially different blood plaama ~amples are shown. For the purposes of the pre~ent invention, the spectral peaks produced by methylene (CH2) and methyl ~CH3) protona are required and they appear in the chemlcal sh1ft spectral reglon o~ l.33 to 0.70 ppm whlch 19 shown along the horlzontal axls. Each spectral peak 19 produced by the arlthmetlc sum of four NMR slgnals produced by the blood plaama constltuents VLDL, LDL, NDL and proteins. It can be seen that the llneshape of the whole plasma ~pectrum ls altered substantially by the change in relative amounts of lts four llpoproteln constituents. However, the llneshapes of the four lipoproteln constltuenta remaln substantlally the same, deaplte the fact that thelr amplltudes change dramatlcally with their relative concentratlons ln the plasma sample. It 19 the lnvarlant llneshape of the NMR qpectra of the lour plasma 11poproteln constltuents across the entlre populatlon and the ... ... .. .... .... .... ............. .......... ...... ... ........ ... . ..

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,-fact that these lineshapes may be arithmetlcally added to produce the lineshape ~f the blood plasma sample, which is the basis for the present lnvention.
Slnce the observed CH2 and CH3 lineshapes of whole plasma samples are closely simulated by the appropriately weighted sum of llpid signals of its four llpoprotein constituents, it is possible to extract the concentrations of these constituents present in any sample. This i~ accomplished by calculating the weighting factors which give the best fit between observed blood plasma NMR spectra and the calculated blood plasma spectra. The process of UMR lipoprotqin analysis is thu3 comprised of the following steps: l) acquisition of an NMR
"reference" ~pectrum for each of the four pure plasma constituents (VLDL, LDL, HDL, Protein), 2) acquiaition of whole pla~ma NMR spectra using measurement conditlon~ identical to those used to obtain the reference spectra, and 3) computer deconvolution of the plasma NMR spectra ln terms of the four constituents to give the concentration of each lipoprotein constltuent expressed as a multlple of the concentratlon of the corresponding lipoprotein reference. The plasma lineshape analysls is accomplished by calculatlng welghtlng coefflcients for each of the four reference NMR spectra whlch minimize the sum of squared deviations between the obaerved plaama NMR
spectrum and that whlch 19 calculated by summlng the four weighted reference spectra.
Whlle the lnventlon 1~ descrlbed hereln as belng used to analyze blood plasma, lt can also be used with equal effectlveness to analy~e blood sérum. Also, the accuracy of the analysi~ can be lmproved if non-llpoproteln constituents are taken l~to account. Whlle future development may expand the 11st of such constltuents, the NMR slgnals produced by metabolltes such as lactate, valine and hydroxybutyrate are .

. ' ' ' ' . , ~ - .. ,; . , ' , , slgnlflcant and should be lncluded in the analysis. The contribution of these constltuents to the MMR signal of blood plasma is illustrated ln Flg. 3, and although they are very small when compared to the llpoproteln constituents, they do affect the accuracy of the deconvolution process.

De~cr;~t10~ Oe the Preferred Fmhodiment Blood ls collected from donors who have fasted for 12-16 i hours. This reduces the amount of chylomicra, whose NMR
spectra are similar to VLDL and which is present ln varlable amounts ln non-fasting donors. The blood is drawn lnto a purple-topped Vacutalner tube containlng ethylenediaminetetraacetic acid (EDTA) and it is then immediately placed on ice. The blood sample is centrifuged at 4C for ten minutes at 1,000 ~ g within four hours after being drawn. The separated blood plasma is pipetted off into a plastic tube, and 0.5 ml is transferred to a Smm outside diameter NMR tube. The plasma sample is then refrlgerated at 4C until the NMR analysls is perfbrmed, whlch should be within 48 hours of lts collectlon.
The above procedure 18 used to collect sample plasma for both analysl~ accordlng to the present lnventlon and for analysis ln order to establlsh reference NMR spectra of the four constltuents. As lndlcated above, the reference NMR
spectra are requlred ln order to practlce the psesent lnventlon. As long as the NMR measurement condltlons remain constant, however, the ~ame reference NMR spectra may contlnue to be employed to analyze further blood plasma samples.
She re~erence NMR spectra mu~t first be obtained for each o~ the four constltuents o~ blood plasma: VLDL, LDL, HDL and Proteins. Plasma 1~ obtained as described abovo and sodium azlde ~N~N3) 1~ added to a 30ml ~ample to glve a ooncentration .. . . . . .

13279~3 of 0.05~ by weight. The sample plasma is hen fractionated into four constituents of dlfferent densities by sequential -flotation ultracentrifugatlon at 10C as described by V.N.
Schumaker and D.L. Puppione, "Sequentlal Flotation Ultracentrifugation~ e ~ ~y~ y, Vol. 12a, pp. 155-170, Academic Press, New York, 1986. The four constituents are defined as follows: VLDL (d<1.006 g/ml), LDL (d=1.006 to ; 1.063), HDL ~d=1.063 to 1.21), and ProteLn ~d~1.21). More specifically, the procedure is to divide the plasma into two groups, ~1 and #2. No adjustment is made of the density of #l (d=1.006) and the density of #2 is ad~usted to 1.063 g/ml by addition of the appropriate volume of a concentrated solution of sodium bromide (NaBr). The two groups of plasma are centrifuged ln 2 ml plastic tubes at 50,000 rpm in a Beckman 50.3 Ti ultracentrifuge rotor for 18 hours. The top fraction ; of ~1 containing pure VLDL is removed and stored at 4C. The denqity of the bottom fractlon of ~1 ~contalning LD~, HDL, and Protein) is ad~usted to d - 1.063 (~3) and tbe bottom fraction of ~2 (containlng HDL and Proteln) lq ad~usted to d=1.21 ~4).
These two groups of sampleq are recentrlfuged at 50,000 rpm for 24 hours. The top fractlon of #3 contalns pure LDL, the top fraction of #4 contains HDL, and the bottom fraction of #4 contains Protein.
At this polnt, the solutions of the four separatsd plasma constituents still contain certain small molecular weight metabolltes, whose methyl proton NMR slgnals appear ln the same spectral reglon as the deslred llpld methyl and methylene resonancea. These compoundQ, whlch would lnterfere wlth the llneshape analyqlJ, are removed by repeated ultraflltration of the four component llpoproteln solutlons at 4C ln a Centrlcon 10 mlcroconcentrator manufactured by Amlcon Corp. After each 5-fold concentration qtep, the llpoproteln solutions are ', ~' ~', .'':.

- diluted t~ thelr orlginal concentration wlth a "mock" plasma solutlon of 0.08M NaBr, O.O5M sodium phosphate, 0.005M ~DTA, O.OOlM CaC12, pH 7.4. Aliquots ~0.5ml) of each sample constituent are placed in 5mm NMR tubes and stored at 4C until analy~i3.
The NMR spectra of the four reference lipoprotein constLtuents are now acqulred. They are stored in computer memory and the lineshapes and amplitudes of their methyl and methylene lipld resonances serve as the references used in the lineshape fitting process that is employed to deconvolute blood plasma sa~ples. Slnce the lineshapes and amplitudes of the N~R
spectra depend quite sensitively on the NMR measurement parameters, most notably magnetic field strength and temperature, it is essentlal that the lipoprotein reference spectra be acquired under the same measurement conditlons to be u~ed when measurlng the whole plaama ~amplea.
In the preferred embodiment, the NMR measurements are conducted at 250 MHz using an unmodified commercial spectrometer, model WM250 manufactured by Druker Instrumenta, Inc. A fixed-frequency 5mm lH probe is installed and the ; temperature controller ls set to 23C ~+0.5C). Fleld homogeneity la optimized by shimming on a sample of 99.8~ D2O
I until the spectral linewidth of the HDO NMR signal is less than I O . 6 Hz . The 90 RF excitatlon pulse wldth 19 set to a value of 5.5 ~ 0.2 microseconds for the D2O measurement.
Referrlng particularly to Flg. 4, the speCtrometer indicated by dashed line 10 is controlled by a digital computer 11. The computer 11 is sold under the trade name ~ASPECT 2000"
and lt has a 24-bit word length and storage for 80~ words. It 30 19 partlcularly well sulted for performing fast Fourier transformatlons and lncludes for this purpoae a hard-wlred sine table And ha~dwlred multlply and dlvide circult. It also : : , includes a data link 12 to an external personal computer 13, and a dlrect-memory-access channel 14 which connects to a hard dlsc unit 15.
The digltal computer 11 also includes a set of analog-to-digital converters, digital-to-analog converters and slow device I/O ports which connect through a pulse control &
interface circuit 16 to the operating elements of the spectrometer. These elements include an RF transmltter 17 which produces an RF excitation pulse of the duration, frequency and magnitude directed by the digital computer 11, and an RF power ampllfier 18 which amplifies the pulse and couples it to the RF transmit coil 19 that surrounds sample tube 20. ~he NMR signal produced by the excited sample in the presence of a 5.875 Tesla polarizing magnetlc field produced by lS superconducting magnet 21 is received by a coil 22 and applied to an RF recelver 23. The amplified and filtered NMR signal is demodulated at 24 and the resultlng qyadrature signals are applied to the lnterface clrcult 16 where they are dlgltized and input through the digital computer ll to a file in the disc storage 15.
After the NMR data ls acqulred from the sample in the tube 20, it is processed by the computer 11 to produce another file whlch ls stored ln the dlsc storage 15. Thls second file is a digital representation of the chemical shift spectrum and it ls subsequently read out to the personal computer 13 for I storage in its disc storage 25. Under the direction of a program stored ln lts memory, the personal computer 13 processes the chemlcal shift spectrum ln accordance with the teachings of the present inventlon to prlnt a report which ls iO output to a prlnter 26.
It should be apparent to those s~illed ln the art that the functlons performed by the personal computer 13 and its ~ . 12 .: ~ "' .

--`` - 1327993 separate disc storage 25 may also be lncorporated into the functions performed by the spectrometer'a digltal computer 11.
In such case, the printer 26 ls connected directly to the digital computer ll.
Prlor to their measurement, the 0.5ml reference samples are removed from the refrigerator and allowed to rise to a temperature of 23C for a period of from ten mlnutes to two hours. A sealed coaxial insert (Wilmad, Cat.#WGS-8BL) contalning an external standard used for field-frequency lock and normalization of the plasma signal amplitudes ls placed into each plasma NMR sample tube before the spectrum is run.
The composition of this standard insert is 0.008M TSP (sodium 3-trimethyl [2,2,3,3-2H4] propionate), 0.6mM MnS04, 99.8% D20.
The D2O provldes the field-frequency lock signal and the integrated area of the TSP rè~onance is used to normalize the amplitudes of the plasma lipid reaonance~ to correct for variationa ln ~pectrometer detectlon senaltlvlty. The ~olutlon la doped wlth Mn2~~ to paramagnetically broaden the normally I sharp Tse resonance to make lts lntegrated area lnsensltlve to ~mall dlfferences in field homogeneity and to shorten it~ Tl relaxatlon tlme to a value comparable to those o~ the plia~ma llpld resonances ~200 to 500 mllllseconds). The reference ~ample containing the coaxlal ln~ert ls placed at a deflned depth ln the sample tube and placed ln the spectrometer. The sample 19 ~pun at a rate of 20 Hz. After locking on the D2O
slgnal from the coaxlal ln~ert, a brlef ~himmlng of the z and z2 gradient controls is performed using the NMR slgnal of the plasma water.
The reference spectrum la then acqulred ualng a standard one-pulae aequence preceded by a one aecond aelectlve decoupler preaaturatlon pulse of the strong H20 resonance. A apatlally ~electlve compoalte 90 ob3ervatlon pul~e (90x~90y~90-x~90-Y) -~ - 1327993 is used to minimize water suppression artifacts as described by A. Bax, "A Spatially Selectlve Composite 90 Radlofrequency Pulse", in J. ~agn. ~eson., ~ 142-145 (1985), although a normal 90 pulse also gives satlsfactory results. The following acquisition parameters are used: 240 transients (4 dummy scans), 4K data size, quadrature detection, 2800 Hz spectral width (9.9 to -1.2 ppm), 0.73 sec. acquisition time, 1.0 sec. decoupler presaturation pulse 10.2 watt) at the H2O
frequency, 22 microsecond composite 90 pulse, and constant receiver gain for all spectra. The time-domain spectra (FIDs) ; of the four lipoprotein reference samples are digitized and stored on computer disk.
The reference sample FIDs are processed identlcalIy to give the frequency-domain spectra used for the plasma lineshape flttlng analysls. The processing operations of Fourler transformation, phasing, and basellne correction are accompllshed using the atandard commercial software of the NMR
spectrometer (Bruker "DISNMR" program). The FIDs are Fourler transformed using 16X data points after application of a 1.0 Hz linebroadenlng exponential multlpllcatlon function. All spectra are scaled identlcally. The spectra are then phase corrected to give pure absorptlon mode slgnals.
~, The chemical shlft scales of the four llpoproteln reference spectra cannot be referenced to the Ca-EDTA resonance because the ionlc compositlon of these reference samples is dlfferent than plasma (owing to the ultracentrlfugation proc-ss). The shlfta of the methyl and methylene resonances of the llpoproteins and that of Ca-EDTA have been shown to be dif~erently a~fected by lonlc strength, and systematlc measurement of the magnltude of thls effect has enabled the "real" chemical ~hl~ts of the methyl and methylene resonances of the llpoproteln constltuents ln whole plasma to be .,:, .
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~``` 1327993 - determined. These chemlcal shifts are glven below and are used to reference the shift scales of the four llpoprotPin reference spectra.

Y~, T.DT, ~112L ~Q~
CH2 Shift (ppm) 1.233 1.220 1.1~6 1.235, 1.175 CH3 Shift ~ppm~ 0.839 0.~23 0.796 0.895, 0.843 A llnear baseline correction is then applied to flatten the basellne between 1.8 and -0.2 ppm and the Fourier transformed, phased, and baseline corrected spectra are transferred to a personal computer model ec-AT manufactured by IBM Corporation and stored on lts disk.
The system ls now ready to measure plasma samples. The procedure la virtually the aame as that de~cribed above for measurement of the reference samples. The same NMR
spectrometer is used and it i9 aet up to operate in the ldentlcal fashlon used to acquire the lipoprotein reference spectra. The tlme domaln spectrum (FID) of the plasma sample ls acqulred in the ldentical faahlon as the re~erence apectra and lt 19 processed ln nearly the ldentlcal manner to produce a dlgitlzed repreaentation o the blood plasma sample spectrum ln the dlsk of the personal computer. The only dlfference ln thls proceaslng is that the whole plasma spectra can be accurately referenced to the sharp NMR resonance peak produced by the I calcium complex o~ EDTA which i8 present in the sample. The ,1 25 entlre spectrum la shifted as needed to allgn thls peak at 1.519 ppm on the horlzontal scale.
The personal computer atorea a program which flts the llneshape of the sample plaama spectrum by a welghted linear comblnatlon Or the ~our llpoproteln reference spectra. ~oth the real and lmaglnary parts o~ the apectra are uaed to make the ~lt in order to correct ~or unavoidable amall relatlve '' ' '' ' ' ~ ~ ,''' :'. ', ~, ' ". ' ' .
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- phase differences between the sample plasma spectrum and the lipoproteln reference spectra. Accurate lineshape analysls also depends on correct alignment of the methyl and methylene reglon of the sample plasma spectrum with the same spectral reglon of the four reference spectra (whose relative alignments with respect to one another are fixed). Small chemlcal shlft differences among plasma samples of slightly different ionic composition are compensated for in the program by systematically moving the sample plasma and reference spectra relative to each other one data point at a time to find the minimum root mean square deviation between the actual measured spectrum and the calculated plasma spectrum.
The mathematics used in the lineshape fitting process (i.e. least squares fit of an unknown function in terms of a weighted sum of known functions) is well known and ls described in many textbooks of numerical analysis such as F.9.
Hildebrand, Tntroductlon to Numerica~ Analysis, 2nd edltion, pp. 314-326, 539-567, McGraw-Hill, 1975. A program for performlng thls functlon on a PC-AT computer ls dlsclosed in the Appendi~. The data pointa of the real part of the sample plasma spectrum whlch comprise the spectral region to be flt (normally 1.33-0.70 ppm) are entered lnto an array. Thls plasma array consists of m dlscrete data polnts denoted Pi, 1-1,2,...m. The data polnts of both the real and lmaglnary parts of the four llpoproteln reference apectra for the same spectral reglon are entered lnto separate arrays. The data points of these arrays are denoted V~lR and v~I for the real and lmaglnary parts, respectlvely, where 1-1,2,...m data polnts and ~-l,l,...n constltuents tn-4 lf only the four llpoprotein constltuents are used ln the flt and n-7 lf the three non-llpoproteln constltuents of Flg. 3 are added to the analysis).

.. . , . , , .. ... , ., . .. . .. ....... ~ . .. .

, " ' . ' '.,. ' ' . ' ' ' ' ,. ' ' " :,~, ' . . , . ,. ~ ' ' :
.

. .
:', ~: ~ . ,: :
: . .. ::
.: ~" . '. ~ ~ ' The method for fitting the measured sample plasmaspectrum, Pl, with a llnear combinatlon of n constituent spectra is based on the premise that there are a set of coefficients (weighting factora), c~R and cjI, corresponding to the real and imaginary contributions of component ; to the observed spectrum such that for each data point Pi ~ ~, CjR VjiR + ~ cjI VjiI .~ piC (calculated plaama spect~um) j=l j-l The best fit will be achieved when the root mean square error, ~ m n (~l2) ls minimized, where i = Pi - Pi~ -~his will be accomplished by finding those coefficients which ~ ~i2 minimize ~i2, that ia, when ~ 0, j = 1,2,...2n (n real ac~
plus n imaginary contributions). Differentiation results in 2n aimultaneout~ linear equation~:

m 2n m Pl Vk1 - ~ c; ~ Vkl V~l k - 1,2,2n , . i'l ~-1 1-1 /
If we let i m m ak~ ~ ~ Vkl V~1, and Sk ~ ~ Pl Vk .~ .
then there are 2n t~lmultaneous llnear equation~ of the form:
2n i ak~ ~ Sk, k - 1,2,...2n 20 Formlng the 2n x 2n matrix, lA] - ~ak~], j-1,22n;
~-1,2..... 2n, glves ~A~ t, where ~ and 1~ are the column vectora, ' ; 17 .~ .

;,. ..
i~ , . .

-~` 1327993 --ClR--c2R

~_C~ ~ and The coefficients providing the best fit are calculated by matrix inversion, . ~ = [A] -1 ~p The root mean square deviation (RMSD) ls computed as aRMs ~ (p ~ O_p ~ C ) 2 To compenqate for lmproper alignment of the Rample plasma data array with re~pect to the reference data arrayR, the Rample plaama data set i8 moved one data point at a tlme in both dlrectlonq with recpect to the reference data. New coefflclents and root mean aquare devlatlon~ are calculated for each allgnment to flnd the beQt flt (minimum RMSD).
For each constltuent ~ there haQ thua been calculated a real and lmaginary coefflclent, clR and clI. The net welghting coefflclent for each conatltuent lq, therefore, glven by: .

i, C~ C~R) 2 + ~C~I) 2 and lts phage by 01 - tan~l ~clI/clR~

. .

.

.:', ' ' ~ ' ~ ' ' ~, .

~ 132799~
.
The total phase shlft of the calculated plasma spectrum versus that of the measured plasma spectrum is glven by:

0 = tan ~ C~ ci ) j~l j'l The component coefficlent~ re~ulting from thl~ lineshape S analysis provide the concentration3 of the four lipoproteln constituents in each plasma sample. Each concentration is expressed relative to the concentration of the lipoprotein who~e spectrum ls used a~ the reference. The f~nal concentrations are normalized to the integrated area of the resonance from the TSP external standard to correct for variations in the detection sensitivity of the NMR
spectrometer.
The information derived rom the above procedure, whLch i9 very rapid (mlnutes) and requlreq almost no sample manipulation, i8 equivalent to that provlded by acqulring separate spectra of the four componenta prepared by ultracent~ifugation ~days) and comparlng the integrala of their lipid NMR signals to those of reference lipoprotein samplea.
It is important to note that what ls being measured by this procedure (NMR signal amplltude origlnatlng from the ~mobile"
lipld molecules in each class of lipoprotein) ia related to, but fundamentally dlfferent from, lipoproteln llpld and protein concentrations derived by the varlous chemlcal and lmmunochemical assays in current clinical use. There is thus no reason to expect a perfect correlatlon to exlst between these NMR-derlved llpoproteln levels and thoqe derlved from standard serum chole~terol and trlglycerlde analyse~. Despite well documented limltatlona ln the accuracy and precialon of the latter measurementa, they are ln wldespread cllnlcal use because of thelr proven val~e ln assesslng coronary heart ..
.. : :
: . ..
- : :.-:

~327993 disease ris~ and other llpid-related dlsease states. It ls posslble~that llpoproteln levels deri~ed from the NMR lineshape - deconvolutlon process may have even greater dlagnostic utllity, but this wlll not be Xnown until extenslve cllnlcal correlation studies have been performed.
To address the questlon of whether the lineshape deconvolution process gives an accurate indication of the - concentrations of the plasma constituents, I have analyzed a series of artificial plasma samples prepared by mixing together defined quantities of the four lipoprotein constituents. As shown in Table 2 below, good agreement is obtained between the known concentrations o~ the four constituents in each sample and those calculated ~y the computer lineshape deconvolution process.

Tahle ?
- Deconvolutlon of ~nown Plasma Spectra at 250 MHz VLDL LDL HDL PROTEIN
~ , Am~le ~rt t`ho~ Calc True Calc True CAlc True t~ 'rrue.
,i 20 FPL5133 116 .23 .25 .25 .25 .23 .25 .26 .25 FPL6 91 112 .16 .15 .26 .25 .23 .25 .31 .25 FPL7124 31 .25 .25 .20 .lS .23 .25 .33 .25 FPL3131 108 .24 .25 .27 .25 .17 .15 .26 .25 , .
FP~9122 73 .24 .25 .16 .15 ,16 ,15 .2a .25 j 25 Plasma ~amples were prepared by mlxing together deflned volumes of bu~ered sallne solutlon and concentrated stock aolutions o~ VLDL, LD~, HDL, and Protein ~bottom lraction) lsolated by ultracentrlfugatlon o~ pooled plaama from several donors. 250 MHz spectra (240 scans, 1 sec. preaaturation) were taken at 23 o~ both the known plasma samples and the stock llpoproteln solutlons and were processed wlth a 1 Hz . . .

- :.

- :, .. - : ;. :

-`` 1327993 linebroadening wlndow function and basellne flattened between l.~ and -0.2 ppm. After transfer of the spectra to a PC-AT
- computer, lineshape analysis was performed on the region of the spectrum contalning the methyl and methylene lipid signals of S the known plasma spectra by deconvoluting the signal as described above in terms of the amplitudes of the signals of the 4 lipoprotein constituents present in each plasma sample expressed relative to the concentratlons of the stock lipoprotein solutions whose spectral amplitudes serve as -reference standards. Table 2 presents the componentlipoprotein concentrations calculated by lineshape analysis (Calc) compared to the true, known concentrations ~True) in the different plasma samples. Triglyceride (TG) and total cholesterol (Chol) contents of the four reference lipoproteins were dlrectly assayed and thè lipid contents of the plasma samples were calculated based on thelr known lipoprotein compositlons.
As indlcated above, the accuracy of the method can be lmproved by conslderlng other blood plasma constltuents ln the analysls. More speclflcally, the metabolltes: lactate, valine, and hydroxybutyrate produce small, but dlscernable proton NMR signals as lllustrated ln Flg. 3. As with the four lipoproteln constltuents, these metabolite constituents can be separated and reference NMR spectra produced for each. These addltional constituents are then added to the deconvolution proce~s aa described above to more accurately determine the concentratlons of the lipoprotein constituents ln the sample blood plasma.
It should be apparent to those akllled in the art that many variations are possible from the above-described preferred embodlment o~ the lnvention. For example, the polarlzlng fleld strength may be lncreased to further qpread the NMR spectrum ' ' : '' . ' : ' ' . . .
.

~ ` , ~ " ' - , ' ', ' - `- 1327993 and to thereby improve the resolution of the deconvolution process. Also, the measurements may be conducted at other temperatures. RegardleSs of the magnetlc fleld strength or the measurement temperature whlch ls chosen, lt is important that 5 the chosen values remain constant throughout the process of - producing the reference spectra and the sample spectra.., .

f 22 :, :

. . . . . . . .

APPENDIX
DEFDBL A-H,O-Z
DEFINT I-N
DIM MSG$(20),RCS(20) DIM YLABS(ll),LPP(6),LPQS(6) COMMON SHARED /MESS/ MSG$(),RCS(),RETS,NRET,NLAG
COMMON SHARED /PRNT/ PICS,ELIS,CMPS~FFS,NPT,NPCT~NPG
COMMON S~ARED /ALPHl/ XTLT,CLS~CR$~LFS~ECOS,QOS,COS,XTIM
COMMON SHARED /ONE/ PL(l),VLD(l),FLD(l),HDL(l),PRO(l),PAT(l),pTR(l) ; COMMON SHARED /TWO/ AM(2),AI(2),C(l),S(l),P(l),A(l),M(l),L(l),PLT(l) COMMON SHARED /TRE/ VIM(l),VLR(l),VIR(l),FIM(l),FIRtl),PRM(l) COMMON SHARED /FOU/ FLR(l),H~M(l),HDR(l~,HIR(l),PRP(l),P~R(l),PIR(l) C0MMON SHARED /FIV/ Jcs(l),Jcss(l),AMs(2),ccON(l),PIN(l),PIM(l),FILS
COMMON SHARED /SIX/ Z(2),SE(l) FILS=''NONE!!'' LPQ$(1)="EXPTL. PLASMA"
LPQS( 2 )="CALCD. PLASMA"
LPQ$(3)="VLDL"
LPQS(4)="LDL"
LPQS(5)=''HDL'~
LPQS(6)="PROTEINl' COLOR 15,1:CLS
MSGS(l)=" PLFIT"
MSGS(2)=" "
MSGS(3)=''NMR LIPOPROTEIN ANALYSIS "
MSG$(4)=" "
MSGS ( S ) =
MSGS(6)~"ENTER RETURN ~0) FOR PARAMETERS TO USE DEFAULT VALUES!"
MSG$~7)-" "
MSGS(8)=
MSGS(9)'"DENNIS w. aENNETT"
MSGS(10)-"1988 VERSION"
CALL MESSAG
SCLMX~-100000:SCLMN-100000 KK-5 'KK ' NUMBER OF PARAMETERS TO V M Y -- C(1)...... C(K) INPR'0:BLIN5''''':LSP~23:CO$-CHRS(58):US-CHRs(24):Ds-CHRs(25) : BKS~" ":FOR I-l T0 79:BLINS=BLINS~BK$:NEXT I
BKINS'" ";Qo$=CHRSt34) BLK$~"
PlX-1122:PIY~1000:P2X=9763:P2Y-6466:NSPLTDl : XT$-"XT;":YTS-"YT;":CPS-"CP":LBS-"LB":IPS-"IP":VS$-"VS"
SPS-"SP":INS-"IN":SCS~"Sc":ITS=cHRS(59):LTS~cNRS(3) CS-",":PAS'"PA,":PRS'"PR,":PUS~"PU,":PDS-"PD,~:TLS-"TL"
j XMN-0:XMX'10:YMN-0:YMX-10 DEF FNXYS(X,Y)-STRS~X)+cS+STRS~Y) DEF FNNS(N)-RIG8TS~STRS(N),LEN(STRS(N))-l)+CS
DEF FNPUS(X,Y)-PAS+PUS~FNXYS(X,Y)+ITS
DEF FNPDS(X,Y)~PAS+PDS+FNXYS~X,Y)~IT$
Plxs~FNN$(plx):plys-FNNs(ply):p2xs~FNNs(p2x):p2ys~FNNs(p2y) XMNS-FNNS~xMN):xMxs~FNNs(xMx):yMNs~FNNs(yMN):yMxs~FNNs~yMx) : NVIN-l:NLIN-l:NHIN-l:NPIN'l CLOSE yl:OPEN "PLPCON" FOR APPEND AS #l:CLOSE #l OPEN "PLPCON~ FOR INPUT AS #l IF EOP(1)-0 THEN 101 STD-90000:NLas.45:NRBs_45:NTsp,7240 'TSP INTEGRAL STANDARD & L~R DATA POIN
VLDLS$~''VLDL'':LDLSS~''LDL'':HDLss~'~0DL'~:pRoTss~'lpRoT'~:FILss~'~NoNE!!
NSTOR-6280 'BEGINNING DATA POIUT FOR INPUT DATA
NVEST-6278 ~BECINNING OF DATA READ IN FROM VLDL FILE
NLDST-6279 'aEGINNING OF DATA READ IN FROM LDL FILE
NHDST~6278 'BECINNING OF DATA READ IN FROM HDL FILE
NPRST-6805 'SEGIUNING OF DATA READ IN FQOM PROT FILE

``` 1327933 NMTSl=6280 '~EGINNING OF MET~YLENE REGION IN PLASMA FILE
- NMTS2=6395 'ENDING OF METHYLENE REGION IN PLASMA FILE
NMESl=6543 'BEGINNING OF METHYL REGION IN NZX POINT ARRAY
NMES2=6726 'ENDING OF METHYL REGION IN NZX POINT ARRAY
VD-.01 '8ASELINE ROTATION FACTOR
PCOMS="COM2:9600,5,7,1,RS,C565535,DS,CD"
NZX-1000:XX=5 CLOSE ~l:OPEN "PLPCON" FOR OUTPUT AS #l PRINT #l,VLDLSS
PRINT ~l,LDLSS
PRINT #l,HDLSS
PRINT #l,PROTSS
PRINT #l,FILS$
PQINT #1, Qo$; PCOMS; QS
PRINT #1,NSTOR;NVLST;NLDST;NHDST;NPRST
PRINT #1,NMTSl;NMTS2;NMESl;NMES2 PRINT #l,VD;NZX;K~
PRINT ~l,STDiNL~S;NR~S;NTSP
CLOSE #l 101 INPUT #l,VLDLSS
INP~T #l,LDLS$
INPUT #l,HDLSS
INPUT #l,PROTSS
INPUT #l,FILSS
INPUT ~l,PCOM$
INPUT Yl,NSTOR,NVLST,NLDST,N~DST,NPRST
INPUT #l,NMTSl,NMTS2,NMESl,NMES2 INPUT ~l,VD,NZX,KK
INPUT #l,STD,NLSS,NRLS,NTSP
CLOSE ~1 102 NZQ-NZX+200 '100 POINT BUFFER ON EACH SIDE
NZP~NZX+100:NZ5'500 DIM PL(NZQ),VLD(NZQ),FLD(NZQ),PLT(NZQ) DIM HDL(NZQ),PRO(NZQ),PAT~NZQ),PTR~NZQ) DIM VIM~NZQ),VLR~NZQ),VIR(NZQ),FIM(NZQ),FLR(NZQ),FIR(NZQ) DIM HIM~NZQ),HDR~NZQ),HIR~NZQ),PRR~NZQ),PRM~NZQ),PIR~NZQ) DIM Z~XX,NZ5),AM(KX,XX),AI~KK,XX),C(KK),S~XX),P(NZQ),A~NZQ),M~NZQ),L(NZQ) OIM JCS~XK),JCSS~XX),AMS(XX,XX),CCON(XX),PIN(NZQ),PIM(NZQ),SE(KK) ; 171 CLS
MSGS(l)-~ ENTER OPTION:"
MSGS(2)-'' "
MSGS(3)-"F. CONTINUE WITH PROGRAM --> FIT DATA!II!!
MSGS(4)-"L. SELECT DATA FILE AND LIPOPROTEIN F}LE PARAMETERS."
MSGS~5)'"N. SELECT NON-LIPID FILE PARAMETERS."
MSGS~6)-"A. AUTO-INTEGRATE TSP PEAX ~ COMPUTE CONC. FACTOR."
MSGS(7)-"I. VIEW/INTEGRATE SPECTRUM.
MSG$(8)-"D. VIEW DIRECTORY."
MSGS(9)'"M. MODIFY STANDM D ~ DEFAULT PM AMETERS."
MSG$(10)-"X. EXIT PROGRAM."
NRET-8:RC$(1)-"L":RC5~2)-"N":RCS~3)-"I'':RCS~4)-''D":RC$~5)~''M'' i RCS~6)-"FH:Rc$~7)~"x":Rcs(8)~"A":cALL MESSAG
IF RETS~"XH THEN 778 IF RETS-"A" THEN 119 IF RETS~>"F" THEN 5555 IF FILS<>"NONEII" THEN 6666 MSGS~ "YOU HAVE NOT SELECTED A DATA FILE!I"
CALL MESSAG

S555 IF RET5~>"I" THEN 113 ~327993 NSTE=NSTOR
CALL PLOTPL(NSTE) NDIN=l SCREEN 0:COLOR 15,1:CLS

113 IF RETS~>"D" THEN 114 MsrNs~l~ENTER DRIVE LETTER -->"
INPR=0:CALL SETIN(INPR) CALL INPT(MSINS,ESP) LOCATE 11, LSiP,0:INPUT D~V~
DRV$=LEFTS~DRVS~l):DRvs=DRv$+ll:
SHLS="DIR ''+DRVS+''/P'' SHELL SHLS
LOCATE 24,4s,0:PRINT "Strike a key when ready 1010 As=INxEys:rF AS="" THEN 1010 C~S

114 IF RETS<~''M'' THEN 115 CALL DEFLT
CLOSE #l:OPEN "PLPCON" FOR INPUT AS #l INPUT #l,VLDLSS
rNpuT ~l,LDLSS
INPUT #l,HDLS$
INPUT #l,PROTSS
: INPUT #l,FILXS
INPUT #l,PCOMS
. INPUT #l,NSTOR,NVLST,NLDST,NHDST,NPRST
INPUT #l,NMTSl,NMTS2,NMESl,NMES2 . INPUT ~I,VD,NZX,KK
j INPUT ~l,STD,NLSS,NRBS,NTSP
. CLOSE #l ~ GOTO 171 115 IF RETS-"L" THEN 111 .IF RETS~"N" THEN 112 111 VLDLS=VLDLSS
LDL$-LDLSS
HDLS-HDLSS
PROTS-PROTSS

NSTEN--NSTOR~NZX-l ; NVLND-NVLST+NZX-l NLDND-NLDST+NZX-l ~.. , NHDND~NHDST+NZX-l " NPRND-NPRST+NZX-l $~2~ " FILE DATA --> SELECT LETTER TO MODIFY;"
MSG$~3)-H FILE/DATA NAME START END~
- MSCs~4)~l H
~ FFFS HD. ~+PFF$ ":FFF9'LEFTS(FFF$,24) s MSCS~S)-FFFS+STRS(NSTOR)+" "+ STRS~NSTEN) :1 FFFS~VLDLSS+" ":FFF$'LEFTS~FFF$,24) FFFS-IlV. "+FFF$
MSCS(6)-PFF$+STR$(NVLST)+" "+ STR$~NVLND) FFF$-LDLS$+" '':FFF$~;LEFT$~FFFS,24) FFFS~"L. "+FFFS
MSG$~7)-FFFS+STRS~NLDST)+" "+ STRS ~NLDND) ,, , i ~ , .

~, ., :

1327~93 FFFS=HDLSS+~ ":FFFS-LEFTS(FFFS 24) FFFS="H. "+FFFS
MSGs(8)=FFFs+STRS(NHDsT)+'~ "+ STRS(NHDND) PFFS~PROTSS+" '':FFFS-LEFTS(FFFS/24) FFFS-"P. "+FFFS
MSG$(9)-FFFS+STR$(NPRST)+'' n+ STRS~NPRND) MSGS(lO)~"METHYLENE REGION "+STRS~NMTSl) MSGS(10)-MSGS(10)+" "+ STRS(NMTS2) MSGS(10)-"A. ''+MSGS(IO) MSGS(ll)-"METHYL REGION "+STRS(NMESl) MSGS(ll)-MSGS(ll)+l~ "+ STRS(NMES2) MSGS(ll)-"~. ''+MSGS(Il) VVX-CSNG(VD) MSGS(12)="N. NON-LIPID PARAMETERS."
MSGS(13)="C. CONTINUE -- SELECTIONS COMPLETE."
NRET=9:RCS(l)=''D'':RCS~2)=''v'':Rc$(3)=''L'':RCs(4)=''H'':RcS(5)=l'P'' RCS(6)='~A~:RC$(7)=~8~:RC$(8)=~C~:RCS(9)=~N~:cALL MESSAG
IF RET$="N" THEN 112 IF RETS="C" THEN 171 IP RET$~>"D" THEN 6659 NDIN-l MSINS=''ENTER NAME OF PLASMA DATA FILE:"
INPR-0:CALL SETIN(INPR) CALL INPT(MSINS,LSP) CURS-PILSS:GOSU~ 2~45 LOCATE ll,LSP,0:INPUT FILS
IF FILS-"" THEN FIL$-FILS$
FILSS'FILS
CLS
MSIN$-"ENTER STMTINC DATA POINT # FOR PLASMA DATA:"
INPR-0:CALL SETIN~IUPR) CALL INPT(MSIN$,LSP) CUR$-STRS(NsToR):GOSUB 2345 LOCATE ll,LSP,O:INPUT NSTR
CLS

NSTOR~NSTR
CLS

6659 IF RET$~>"V" THEN 6658 NVIN-l MSIN5-~ENTER NAME OF VLDL COMPONENT FILE-"
INPR-O:CALL SETIN(rNPR) CALL INPT~MSIN$,LSP) 6 CUR$-VLDLsS:GoSUB 2345 LOCATE ll,LSP,0:INPUT VLDLS
IF VLDLs-"H THEN VLDLS-VLDLSS
VLDLSS-VLDLS
CLS
MSINS-''ENTER STARTING DATA POINT ~ FOR VLDL DATA:"
INPR-O:CALL SETIN~INPR) CALL rNPT~MSINS~LSP) CURS-STRS~NVLgT):GOSUS 2~45 LOCAT~ ll,LSP~0:INPUT NVSTR

IF NVSTR-O THEN NVSTR~NVLST

i .

, NVLST=NVSTR

6658 IF RET$~>~L~ THEN 6657 NLIN=1 MSIN~-"ENTER NAME OF LDL COMPONENT FILE:"
INPR=O:CALL SETIN(INPR) LSP=2 3 CALL INPT(MSIN$,LSP) CURS~LDLS$:GOSUB 2345 LOCATE 11,LSP,0:INPUT LDLS
IF LDL$="" THEN LDLS=LDLSS
LDLSS=LDLS
CLS
MSINS=~ENTER STA~TING DATA POINT # FOR LDL DATA:"
INPR=O:CALL SETIN~INPR) LSP=19 CALL INPT(MSIN~,LSP) CURS--STRS(NLDS$):GOSU~ 2345 LOCATE 11,LSP,O:INPUT NLSTR
CLS
IF NLSTP~=O THEN NLSTR=NLDST
NLDST=NLSTR

6657 IF RETS<>"H" THEN 6656 MSINS~ENTER NAME OF HDL COMPONENT FILE:"
INPR=O:CALL SETIN(INPR) LSP~22 CALL INPT(MSINS~LSP) CURS'HDLSS:GOSU8 23 4 5 LOCATE 11,LSP,O:INPUT HDLS
IF HDLS ~"" THEN HDLS~HDLSS
HDLS$~-HDLS
CLS
MSINS~"ENTER STARTING DATA POINT # FOR HDL DATA:~
INPR'O:CALL SETIN(IN2R) CALL INPT~MSIN$,LSP) CURS~STR$~NNDST):GOSUB 2~45 LOCATE 11,LSP,O:INPUT NHSTR
CLS
IF NHSTR~O THEN NHSTR-NHDST
NHDST--NHSTR

6656 IF RETS~>~P~ THEN 6655 , MSIUS'~'ENTER UAME OF PROTEIN COMPONENT FILE:"
INPR--O:CALL SETIN~INPR) LSP~21 CALL INPT~MSINS,LSP) CURS~PROTSS:GOSUB 2 3 4 5 LOCATE S1,LSP,O: INPUT PROTS
IF PROT$~ THEU PROT$--PROTSS
PROTSS--PROT$
CLS -MSINS--"ENTER STARTING DATA POINT # FOR PROTEIN DATA:I~
INPR~O:CALL SETIN~INPR) LSP~17 CALL INPT~MSINS~LSP~
CURS~STR$~NPRST):GOSU3 2345 . ~ . .. - r :: :

1327~33 LOCATE ll,LSP,0:INPUT NPSTR
CLS
IF NPSTR=0 THEN NPSTR=NPRST

6655 IF RETS<~"~" THEN 6654 NAIN~l MSINS~"ENTER STARTING DATA POINT FOR METHYL REGION:I' INPR=O:CALL SETIN(INPR) LSP~18 CALL INPT(MSINS,LSP) CURS-STRS(NMESl):GOSU~ 2345 LOCATE ll,LSP,0:INPUT NMEl CLS
MSINS="ENTER ENDING DATA POINT FOR MET~YL REGION:"
INPR=0:CALL SETIN(INPR) LSP-l9 CALL INPT(MSINS,LSP) CURS=STR5(NMEs2):GoSUB 2345 LOCATE ll,LSP,0:INPUT NME2 IF NMEl=O THEN NMEl=NMESl IF NME2=0 THEN NME2=NMES2 NMESl=NMEl NMES2=NME2 CLS

6654 IF RETS~>"A" THEN 111 MSINS-"ENTER STARTING DATA POINT FOR METHYLENE REGION-"
INPR=O:CALL SETIN(INPR) ,, CALL INPT(MSINS,LSP) CURS-STRS~NMTSl):GOSUB 2345 LOCATE ll,LSP,O:INPUT NMTl CLS
MSINS-"ENTER ENDING DATA POINT FOR METHYLENE REGION-"
s INPR'O:CALL SETIN(INPR) LSP'17 CALL INPT(MSINS,LSP) CURS'STRS~NMTS2):GOSUB 2345 LOCATE 11,LSP,O:INPUT NMT2 IF NMTl-0 THEN NMTl'NMTSl IF NMT2-0 THEN NMT2~NMTS2 NMTSl-NMTl NMTS2'NMT2 CLS

112 'MENU FOR NON-LIPID PARAMETERS
'INCLUDE L. --~ 111 : GOTO 171 119 IP FIL$~"NONEII" THEN 1616 MSGS(l)~"YOU HAVE NOT SELECTED A DATA FILEI!N
CALL MESSAG

MSINS-"ENTER NAME OF FILE CONTAINING TSP PEAX --~"
INPR-0:CAL~ SETIN(INPR) LSP~l9 CALL INPT~MSIN$,LSP) ~OCATE ll,LSP,O:INPUT FILIS
IP FILIS-"" THEN FILIS-FIL$

. . .: , .
, : .
' : , ' .

--` 1327993 CLS
NSCR=O
GOSU~ 1617 ~ GOTO 171 1617 CLOSE ~l:OPEN FILIS AS ~1 LEN~4 FIELD 1~4 AS s$
NEND=NSTR+NZQ-l Nl=NST+NSTR*2:N2=NST+NEND*2 X--O
FOR I-Nl TO N2 STEP 2 K~K+l GET l,I
GOSU~ 300 PIN(R)=V/1000 NEXT I
CLOSE #l NTOP=NTSP-NSTR
NLF=NTOP-150:NRT=NTOP~150 TMAX=-100000 : FOR K=NLF TO NRT
IF PIN(K)~TMAX THEN 69 ; TMAX=PIN(X) ; NTOP=K

IPXl=NTOP-NLBS:IPX2=NTOP+NR8S
TENS2-lOO~PIN(IPX2):TENSl=lOO~PIN(IPXl) SLP-(TENS2-TENSl)/(IPX2-IPXl) B=TENSl-(SLP~IPXl) : FOR I-IPXl TO IPX2-1 J~I+l 81- (SLP~I ) +~
1~2' ~SLP~J) +B
Hl-lOO~PIN(I)-Bl H2-lOO*PIN(J)-~l HR-Hl:IF H2cHl THEN HR-H2 AT-APS(H2-Hl):AT~AT/2 AR-HR+AT
; M PX-ARPK+AR
NEXT I
RNORM'STD/ARPK
IF NSCR~>O THEN RETURN
ARPS-STRS(ARPK) LDP-INSTR(ARPS~
M P$-LEFT$(ARP$~LDP-l) MSCSIl) 'TSP PEAX INTEGRATED FOR "+FILIS
. MSGS~3)~"INTEGaAL ~"+ARPS
MSG$(4)-" n MSCS(5)-"TOP OF PEAK LOCATED AT"+STR$~NSTR+NToP) MSGS~6)-"PEAK INTEGRATED FROM"+STR$(NSTR+IPXl)+" TO"+STRS(NSTR+IPX2) MSG$(7)'"INTEGRAL NORMALIZATION FACTOR '"

LOCATE 14,56,0:PRINT USING "#~.#J#";RNORM;
LOCATE 24~28~0:PRINT "PRESS ANY XEY TO CONTINUE":
6497 A$~INKEY$:IF AS-"" THEN 6497 . RETURN
234S LOCATE ~,22,0:PRINT "PRESS RETU~N TO RETAIN ----> I~;cuRs .
.
`:

6666 MsGstl)3l~Do YOU INTEND TO USE T~E PLOTTER?"
MSG$(3)-" Y. YES N NO"
- NRET-2 RCS~l)="Y":RCSt2)~"N":cALL MESSAG
IF RETS-''N'' THEN 6661 NPLOT-l MSGS(l)~"TURN PLOTTER ON AND INSTALL PAPER!"
CLOSE #l:oPEN PCOMS AS #l PLoTs~rNs+ITs+Ips+plxs+plys+p2xs+p2ys+ITs PRINT #l,PLOTS
PLOTS-"PS"+"10"+ITS 'INITIALIZE
PRINT #l,PLOTS 'PAPER SIZE = 8.5 X 11 PLOTS=sPS+FNN$(1)+ITS:PRINT #l,PLOTS
PLOTS=VS5+"9.5,":PRINT #l,PLOTS
PRINT #l,PLOTS 'SELECT PEN VELOCITY
PRINT #l,"SP~"
CLOSE #l 'STORE PEN
6661 MSGStl)-"DO You INTEND TO USE THE PRINTE~?"
~' MSGS~3)='' Y. YES N NO"
NRET=2 RC S (l)=''Y'':RCS(2)=''N'':CALL MESSAG
IF RET$="N" THEN 6662 NPRNT=l MSGS(l)="TURN PRINTER ON AND ALIGN PAPER IF NECESSARY!"
6662 IF NVIN=0 THEN 9090 CLOSE #1 OPEN VLDLS AS #1 LEN~4 NST-641:STOR~0 ' Nl-NST+NVLST~2-200:N2-NsT+NVLND~2+200 - FOR I-Nl TO N2 STEP 2 X-K+l GET l,I

~ VED~K)-V/1000 : VLR(K) V/1000 GET l,I+l .VIM(KJ3V/1000 'IMAGINARY PART OF SPECTRUM
(KX)'V/looo 'IMAGINARY PART OF SPECTRUM
NEXT I
9090 IF NLIN'0 THEN 9089 CLOSE ~1 OPEN LDLS AS ~1 LEN 4 ' Nl-NST+NLDST~2-200:N2.NST~NLDND~2+200 ;~ FOR I-Nl TO N2 STEP 2 ~ X-K+l 7 GET l,I

FLD~X)-V/1000 FLR(X)~V/1000 .. .. .

; ' ' ' ' ' .
.; ' ' , . .

' , ' --` 13279~3 - GET l,I+l ; GOSUB 300 FIM(X)=V/1000 'IMAGINARY PART OF SPECTRUM
FIR(X)~V/1000 'IMAGINARY PART OF SPECTRUM
NLD-K
NEXT I
9089 IF NHIN=0 T~EN 9097 CLOSE #l:OPEN HDLS AS #1 LEN=4 FIELD 1,4 AS SS
Nl-NsT+NHDST~2-200:N2=NST+NHDND~2+200 FOR I=Nl TO N2 STEP 2 K=K+l GET 1, I

HDL(K)=V/1000 HDR(K)=V/1000 GET l,I+l HIM(K)=V/1000 'IMAGINARY PART OF SPECTRU~
NHD(K)=v/looo 'IMAGINARY PART OF SPECTRUM
NEXT I
9097 IF NPIN=0 THEN 1112 CL0SE #l:OPEN PROTS AS #1 LEN=4 FIELD 1,4 AS SS
Nl~NST+NPRST~2-200:N2=NST+NPRND~2+200 FOR I=Nl TO N2 STEP 2 X~X+l GET l,I

PRO(R)-V/1000 PRR~X)'V/1000 GET l,I~l PRM(~) -V/1000 'IMAGIN M Y PART OF SPECTRUM
PIR~X)3V/1000 'IMAGINARY PART OF SPECTRUM
NPO-K
NEXT I
1112 NMTl-NMTSl-NVLST+101:NMT2-NMTS2-NVLST+101 NMEl-NMESl-NVLST+101:NME2-NMES2-NVLST+101 FOR I~l TO KK:JCS~ 0:NSX$ I

' NREG-3 499 MSGS(I~-"SELECT OPTIONS ~ CU~RSNT OPTION)."
MSG$(2)-" "
- MSG$~3)-"A.`FIT METHYL REGION"
IF NREG~l THEN MSG$~3)'"A. FIT METHYL REGION ~"
MSGS(4)-"g. FIT METHYLENE REGION"
IF NREG-2 THEN MSGS(4)-"8. FIT METHYLENE REGION ~"
MSGS~5)-"C. FIT ~OTH REGIONS"
j IF NREG'3 THEN MSGS~5)~"C. FIT BOTH REGIONS ~"
MSGS~6)-"D. CONSTRAIN COMPONENT~S)~
IF NCN-l THEN MSGS~6)-"D. CONSTRAIN COMPONENT~S) ~"
MSGS~7)-"Z. FIT DATA~
MSGS~8)-~R. RET~N TO MAIN MENU"
NRET-6:RCS~l)-"A":RCS~Z)-"B":RCS~3)-"C":RCS~4)-"DN:RCS~S)-"Z"

,, .

~ ' ~
" :, ::.

RC$(6)-"R"
CALL MESSAG
IF RET$=~R~I THEN 171 IF RET$<>11A~I THEN 401 NREG-1:GOTO 499 401 IF RETS~>"8" THEN 402 NREG=2:GOTO 4 9 9 402 IF RET$<>~C~ THEN 403 NREG~3:GOTO 49 9 403 IF RETS<>"D" THEN 411 NCN=1-NCN:GOTO 499 411 NPSC=0 IF NCN=O THEN 234 349 MSGS~ "WHICH COMPONENT CONCENTRATIONS DO YOU WISH TO C0NST~AIN?"
MSG$(3)=" V. VLDL~
IF JCS(1)<>0 THEN MSGS (3 ) -" V. VLDL *~
MSG$(4)=" L. LDL"
IF JCS(2)<>0 THEN MSGS(4)=~ L. LDL *~
MSG$(5)=" H. HDL~
IF JCS(3)~>0 THEN MSGS(5)=~ H. HDL *~
MSGS(6)=I~ P. PROTEIN~
IF JCS(4)<>0 THEN MSGS(6)=~ P. PROTEIN
MSGS(7)=I~ A. NO CONSTRAINTS~
MSGS(8)~ C. CONTINUE --> SELECTIONS COMPLETE"
NRET=6:RCS~1)=''V'~:RCS(2)~'~L'~:RCS(3)=I~H~:RCS~4)='~P'~
RCS(5)="A":RCS(6)~1~C":CALL MESSAG-IF RETS~C'~ THEN 339 IF RETS<>'~V~ THEN 341 JCS~1)-1:JCS(5)=1:GOTO 349 : 341 IF RETS~>"L" THEN 342 JCS~2)-1:JCS(6)=1:GOTO 349 342 IF RET$<>1~H~ THEN 343 JCS(3)-1:JCS(7)-1:GOTO 349 ~43 IF RETSC>~P~ THEN 344 JCS(4)-1:JCS~8)-1:GOTO 349 344 FOR I~1 TO KX:JCS(I)-0:NEXT I

339 F0R I~1 TO KX
IF JCS(I)~0 THEN 369 IF I-1 THEN CPTS'"VLDL"
IF I~2 THEN CPTS~LDLI~
IF I~3 THEN CPTS~IIHDL'~
IF I-4 THEN CPT$-"PROTEIN"
CSSS-STRS(CCON(I)) , MSINS~ENTER VALUE OF '~+CPTS+I~ TO FIX (ENTER --> '~+CSSS+I')~
INPR-0:CALL SETIN(INPR) CALL INPT(MSINS~LSP) LOCATE 11,LSP,0:INPUT CSS
IF CSS'~n THEN C(I)-CCON(I) ELSE C(I)-VAL~CS$) IF CSS.~ THEN C~I+4)~CCON(I+4) ELSE C~I)~0.0 234 IF NDIN'0 THEN 238 IF FILS~ NONEI !" THEN 246 MSCS~ DATA FILE MUST BE SPECIFIEDI ! "
CALL MESSAC

. .

,,.~; i ' ~,"!,1 .. . . . .
: . . ' . ' : ' .
' 246 CL0SE #1:OPEN FIES AS #1 LEN=4 FIELD 1,4 AS SS
NEND=NSTR+NZX-1 N1-NST+NSTR*2-200:N2=NST+NEND~2+200 K=0 FOR I=N1 TO N2 STEP 2 R=X+1 :. . GET 1,I

PAT(X)=V/1000 : PTR~R)-V/1000 NEXT I
C~OSE #1 PPMX=-100000 FOR I=101 TO NZP
IE PAT(I)>PPMX THEN PPMX=PAT~
NEXT I
~MSMN=1000000 ' ~**~*****~* OPTIMIZE BASELINE POSITION --> 1 DATA PT~***********
238 MSG$~1)="FITTING DATA!":CALL MESAG
NMQ1=NME1:NMQ2=NME2:NMR1=NMT1:NMR2=NMT2 NMX1=NME1:NMX2=NME2:NMY1=NMT1:NMY2=NMT2 LOCATE 18,26,0:PRINT "RMSD --> ";:PRINT USING " . #Y#Y#YY###YY";RMS
2011 K'JP-1 K~X+1 IF K<1 THEN 2012 : IF K>NZP THEN 2012 VLD~I)=VLR(X):VIM(I)-VIR(X) FLD(I)3FLR(K):FIM(I)~FIR(K) HDL(I)-HDR(K):HIM~ HIR(X) PRO~I)~PRR(X):PRM(I)'PIR(X) LOCATE 18;26,0:PRINT "RMSD --> ";:PRINT USING " . ###Y~###";~MS
. NME1'NMX1-DLTA:NME2-NMX2-DLTA:lJ~T1=NMY1-DLTA:NMT2=NMY2 DLTA

LOCATE 18,26,0:PRINT "RMSD --> ";:PRINT USING " . Y#YY~#YY";RMs IF RMS>RMSMN THEN 2013 RMSMN-RMS:JST~JP:JP-JP+1 .i GOTO 2011 ;j 2014 X-JP-1 FOR I'51 TO NZP
. X-X+1 IF X~1 THEN 2015 .l IF R>NZP THEN 2015 VLD(I)'VLR(K):VIM~ VIR~X) ` FLD(I)-FLR(X):FIM(I)~PIR(R) HDL(I)-NDR~X):HIM(I)-HIR(X) PRO(I)'PRR~K):PRM~ PIR~X) LOCATE 18,26,0:PRINT "RMSD --> ";:PRINT. USINC "###~.##~##~###~ ";RMS
. DLTA-JP-51 -i NME1-NMX1-DLTA:NME2-NMX2-DLTA:NMT1~NMY1-DLTA:NMT2WNMY2-DLTA
: GOSUD 2000 LOCATE 18,26,0:PRINT "RMSD --> ";:PRINT USING " .##~##YY#Y XY";RMS
IF RMS~RMSMN TNEN 2016 .

:1 , 33 J

' ., , I .

~' ' '. . ' . . . ' , - ~ ' ~` ' . ' RMSMN=RMS:JST=JP:JP--JP-l 2016 X=JST-l RMS~RMSMN
FOR I=51 TO NZP
K=K+l IF K<l T~EN 2020 IF K>NZP THEN 2020 VLD(I)~VLR(X):VIM(I)=VIR(K) FLD(I)-FLR(K):FIM~ FIR(K) ; HDL(I)=HDR(K):HIM(I)-HIR(K) PRO(I)=PRR(K):PRM(I)=PIR(K) DLTA=JST-51 NMEI-NMXl-DLTA:NME2=NMX2-DLTA:NMTl=NMYl-DLTA:NMT2=NMY2-DLTA

2000 II=O
531 PATMX=-100000:PATMN=100000 IF NREG~2 THEN 20Ql FOR-I=NMTl TO NMT2 IF PAT(I)~PATMX THEN PATMX=PAT(I) II-II~l Z(5~II)=vIM(I)(z(6 )I)=F(M)I)Z(z(7I)=FLD(I) Zt3~ I)=HDL(I) Z(4~II)=pRo(I) IF NREG~>3 THEN 2002 2001 FOR I-NMEl TO NME2 IF PAT~I)>PATMX THEN PATMX=PAT ( I) II-II+l zP(5I)I)PAv(I(-Z(l,II)-V~D(I) Z(2, I)-FLD(I) Z(3,II)-HDL~ Z(4 II)-PRO(I) 2002 NZ-II 'NZ - NUMBER OF POINTS TO FIT
LEAST SQU M ES FIT OF LINEAR COMBINATION OF COMPONENTS
IF JCS~J)~0 THEN 719 FOR I~l TO NZ
P~I)'P(I)-C(J)~Z(J~I) NEXT I

MM-O
FOR N'l TO KX
IF JCS~N)<>O THEN 701 MM'MM+ 1 NN-O
FOR J'l TO XK
' IF JCS~J) O O THEN 707 . NN-NN~l j AM~MM,NN)~O
FOR I-l TO NZ
NME~MM~NN)'AM~MM~NN)+z~N~ z~J~I) NN'O
FOR N-l TO XX
IF JCS~N)~0 THEN 703 NN'NN+l S~NN)-0 i . . . ~ .. .. .
. , .. ,, . . , . .. , ,~.

- ~ . .

. ., ' ..
.

FOR I=l TO NZ
s(NN)=s(NN~+p(I)~z(N~I) NEXT I

INVERT MATRIX AM --> AI
N~-NN
CALL INVERT(IE,NQ) IF IE<>0 TNEN 577 CLS
MSGS (1)3"5INGUI~ MA'rRIX! ! "
CALL MESSAG
RETURN
C-AI~S
__________________________________ 577 FOR I-l TO KK -SE(I)=0 IF JCS (I) <>O THEN 505 C(l)=0:SE(I)=AI(I,I) MM=O
FOR I-l TO KK
IF JCS (I) ~>0 THEN 704 MM=MM+ 1 FOR ~=l TO XK
IF JCS(J)~>0 THEN 716 NN~NN+l C~ C~I)+AI(MM,NN)*S(NN) II~0 FOR I=51 TO NZP:PL~I)-0:NEXT I
FOR I-NMTl TO NMT2 I$aII+l FOR J-l TO KK
PL~I)~PL~I)+C(J)~Z(J,II) NEXT J
NEXT I
FOR I=NMEl TO NME2 II-II+l FOR J'l TO KK
PL(I)~PL(I)+C(J)~Z(J~
NEXT J
NEXT I
CALCULATE RESIDUAL, ~MS DEVIATION, CORRELATION COEFFICIENT
RES-0:SPT-0:SPL-0:SMl-0:SM2-0:SM3'0 IF NREG~2 THEN 2005 FOR I~UMTl TO UMT2 ' PL~ C~ VED~I)+C(2)~FLD~I)+C(3)~HDL~I)+C~4)~PRO~I) ; PL~ PL~I)+C(5)~VIM(I)+C~6)bFIM~I)+C~7)~HIM~I)+C~a)~PaM~I) ' SPT-SPT+PAT(I):SPL~SPL+PL(I) DEL~PL~ PAT~I):RES-RES+DEL~DEL
. NEXT I
IP NREG~3 TNEN 2006 2005 FOR I-NMEl TO NME2 PL~ C(l)~VLD(I)+C(2)~FLD~I)+C~3)~HDL(I)+C(4)~PRO(I) PL(I)'PL~I)+C(5)~VIM~I)+C~6)bFIM~I)+C~7)~HIM~I)+C~8)~PaM~I) SPT-SPT+PAT~I):SPL-SPL+PL~I) DELJPL~I)-PAT(I):RES-RES+DEL~DEL
NEXT I
.

- ` .. ..... ........

- . . ~ .
.

`- 1327993 20 06 VRc=REs/tNz) RMS=SQR(VRc)' 'ROOT MEAN SQU M E DEVIATION
PVT=SPT/NZ:PVI~SPL/NZ
IF NREG<2 THEN .005.
FOR I-NMTl TO NMT2 ; SMl=SMl+((E'AT(I)-PVT)~(PL(I)-PVL)) SM2-SM2+(( PAT(I)-PVT~' 2) SM3=SM3+~(PL(I)-PVAT.) 2) ; NEXT I
I F NREG<> 3 THEN 3006 3005 FOR I-NMEl TO NME2 SMl=SMl+( (PAT(I) -PVT)*(PL(I)-PVL)) SM255M2+( (PAT~I)-PVT) ^2) SM3=SM3+( ~PL(I)-PVL) ^2) NEXT I
3006 SM2=SM2*SM3:SM2=SQR(SM2) CARC=SM1/SM2 S'T=O:CT=O
KK2=KK/2 FOR I=l TO XX2 CT=CT+C(I) ; SE(I)-SE(I)*VRC
~ ST=ST+C( I+XK2~
.: SE( I+~R2)=SE(I+XR2)*VRC
NEXT I
TT=ST/CT: THT=ATN (TT)*(180/3.14159) FOR I-l TO KAK:SE(I)=SQR(SE(I)):NEXT I
` FOR I-l TO. XK2 CI=C(I)~C(I)+C(I+XX2)~C~I+XX2) CIS=SQR~CI) SIGI-(1/(2*CIS))~(SE(I)+SE(I+KK2)) .: SE(I)-SIGI
CT=CT+C(I) SE(I)-SE(I) *VRC
ST-ST+C( I+KK2) SE~ I+KK2) ^^SE(A^.+AKX2)~VRC
NEXT I
- RETURN
- 300 B1 ASC~MIDS(S$,1,1)) B2-ASC( MID$ (S$,2~1)) . B3-ASC(MID$~5$,3,1)) B4 ASC~MIDS~S$,4,1)) gB^-^B4 AND ~AH8O CHECX SIGN BIT ON HIGH BYTE
IF SB~0 THEN 500 NEGATIVE ~ 500 V B1+B2~256+B~*256 2+B4~256^3 RETURN

.~ BC2-B2 XOR 255 BC3^^B3 XOR 255 I BC4^-^B4 XOR 255 BC1 BC1+1CREATE TWOS COMPI,EMENT
-, IF BC1<256 THEN 600 BCl'0 BC2'^eC2+1 IF BC2~256 THEN 600 BC2~0 BC3 BC3+1 IP BC3<256 THEN 600 ~c~o 3C4-BC4+1 . .
., - : .

' .~', '' , ' ~ ' .
, - -` 1327993 600 Bl=Bcl:B2=Bc2:~3=Bc3:84=~c4 V=31+~2*256+B3*256^2+~4*256^3 'A~S(W) V~-V IGET SIGN RIGHT
RETURU
'~**** PLOT *****************~*****
2500 GOSU~ 2000 RMSM~=RMS
NSCR-l FILIS-FIL$
GOSU~ 1617 ARPQ- M PX
NTQP=NTOP
IPQl-IPXl:IPQ2=IPX2 RNQRM=RNORM
PLMX=-100000 FOR I=NMTl TO NMT2 PL(I)=C(l)*VLD(I)+C(2)*FLD(I)+C(3)*HDL(I)+C(4)*PRO(I) PL(I)=PL(I)+C(5)*VIM(I)+C(6)*FIM(I)+C(7)*HIM(I)~C(8)*PRM(I) IF PL(I)>PLMX THEN PLMX=PL(I) NEXT I
; FOR I=NMEl TO NME2 PL(I)=C(l)*VLD(I)+C(2)~FLD(I)+C(3)*HDL(I)+C(4)*PRO(I) PL(I)=PL(I)+C(5)*VIM(I)~C(6)*FIM(I)+C(7)*HIM(I)+C(8) *PRM(I) IF PL(I)>PLMX THEN PLMX=PL(I) NEXT I
SCLMX~PATMX
IF PATMX~PLMX THEN SCLMX=PLMX
CLS
WF-600:WI=50 FRG=WF-WI:RINC5FRG/10 WVG-(WI+WF)/2:WVR~(INT((WVG/.5)+.5))*.5 XEY OFF:CLS:WINDOW (-1,-2.5)-(10.5,10.5) REM ***~ PLOT AXES ****~
LINE (0,-.2)-(0,10~,14 LINE (0,-.2)-(10,-.2),14 FOR I-l TO 9:~INE (I,-.4)-(I,0),2:LINE (I+.5,-.3)-(I+.5,-.1),2 NEXT I:LINE (10,-.4)-(10,0),14:LINE (.5,-.3)-(.5,-.1),14 FOR I'O TO lO:LINE (-.l,I)-(.l,I),2 NEXT I
. LOCATE 6,3,0:PRINT "I";
LOCATE 7,3,0:PRINT "N";
LOCATE 8,3,0:PRINT "T";
LOCATE 9,3,0:PRINT "E";
LOCATE 10,3,0:PRIUT "U";
LOCATE 11,3,0:PRINT "S";
LOCATE 12,3,0:PRINT "I";
LOCATE 13,3,0:PRINT "T"s LOCATE 14,3,0:PRINT "Y";
REM ~ PLOT PEAX ****~*
FOR I-NMTl TO NMT2-l:J-I+l - PXl-5-(~WVR~ RINC) PYl-~lO~PL(I))/SCLMX
PX2~5-((WVR-J)/RINC) PY2-(lO~PL(J))/SCLMX
LINE (PXl,PYl)-(PX2,PY2),10 NEXT I
POR I-NMEl TO NME2-l:J'I+l PXl-5-((WVR-I)/RINC) PYl-(lO~PL(I))/SCLMX

;

- . ., ., : , ; ,.
.

.~ . . .
~. :

:,` .. ..

PX2=5-~NVR-J)/RINC) PY2=~10~PL~J))/SCLMX
LINE (PXl,PYl)-(PX2,PY2),10 NEXT I
FOR I3UMTl TO UMT2-l:J--I+l PX1--5-((WVR-I)/RINC) PYl-(10~PAT(I))/SCLMX
- PX2--5--((WVR-J)/RINC) PY2--(lO~PAT~J))/SCLMX
LINE (PXl,PYl)--~PX2,PY2),12 NEXT I
FOR I=UMEl TO UME2-l:J=I~l PXl=5-~WVR-I)/RINC) PYl--~10~PAT~I))/SCLMX
PX2--5-~WVR-J)/RINC) PY2--~10~PAT~J))/SCI.MX
LINE ~PXl,PYl)-(PX2,PY2),12 NEXT I
IF NPSC<~0 THEN 1020 TRES=0.0 FOR I=N~Tl TO NMT2 PL(I)-C~ VLD~I)+C~2)~FLD~I)+C(3)*HDL(I)+C~4)~PRO(I) PL(I)=PL~I)+C~5)~VIM~I)+C(6)~FI}I(I)+C(7)~HIM(I)+C(8)'1PRM~I) DEL=PL(I)-PAT(I) TRES=TRES+DEL~DEL
NEXT I
FOR I=NME1 TO UME2 PL(I)=C(l)~VLD~I)+C(2)*FLD(I)+C(3)~HDL(I)+C(4)~PRO(I) PL(I)-PL(I)+C~5)~VIM(I)+C(6)~FIM(I)+C(7)~HIM(I)tC(8)1~PRM(I~
DEL=PL(I)-PAT~r):TRES=TRES+DEL*DEL
NEXT I
VRC--TRES/NZ
TRMS-SQR(VRC~ 'TOTAL ROOT MEAN SQUARE DEVIATION
ClPR--C~ C(l)+C(5)~C(5):ClPR--SQRtClPR) C2PR--C(2)~C(2)+C(6)~C(6):C2PR~SQR(C2PR) C3PR-C~3)~C~3)1C~7)~C~7):C3PR--SQR~C3PR) C4PR-C(4)~C~4)+C(8)~C~8):C4PR--SQR~C4PR) 1020 LOCATE 1,35,0:PRINT "VLDL -->";:PRINT USING "##JI.~##";ClPR
LOCATE 2,35,0:PRINT "LDL -->"::PRINT USING N t##.t#~":C2PR
LOCATS 3~35,0:PRINT "HDL --~;:PRINT USING "ll#ll.l~##~;C3PR
LOCATE 4~35,0:PRINT "PROT -->";:PRINT USING "il##.#l~#n;C4PR
LOCATE 5,35,0:PRINT "RMSD --~"::PRINT USING "~1###.#J~";RMS;
:. LOCATE 6,3S,0:PRINT IITSD --~";:PRINT USING " .#l~";TRMS;
LOCATE 7,35,0:PRINT "CORR -->";:PRINT USING "~#. ";CRC
LOCATE 8~35~0:PRINT "PHASE --~";:PRINT USING " .~#";THT
IF NREG-l TNEN E~GN$-"METHYL REGION FIT"
IF NREG--2 THEN RGNS--"METHYLENE REGION FIT"
. IF NREG--3 THEN RGNS--"BOTHREGIONS FIT"
LOCATE 10,35,0:PRINT RGUS;
LOCATE 24,28,0:PRINT "PRESS ANY KEY TO CONTINUE";
777 AS--INKEYS:IFAS-"" THEN 777 7778 FOR I--l TO KX:FOR J--l TO NZ5:Z~I,J)-0:NEXT J:NEXT I
FOR I-l TO NZQ:P(I~--0:A(I)-0:M(I)30:L(I~-0:NEXT I
FOR I--l TO KK:FOR J--l TO KX:AM(I~J)~0;AI~I~J)--0 NEXT J; NEXT
FOR I--l TO KK:S(I)-0:NEXT I 'CHECK
900 SCREEN 0l0;WIDTN 80:COLOR 15~1:CLS
MSGS~l)--" OpTIoNs __>n '~ MSGS(2)-n "
MSGS~3)~1lA. CONTINUE -- FIT NEW DATA.

'~ ' 3~

.. .

,i' :
.
''' ' `, ' ' ' . . ~ ' " ~ ' .

.: - - ,, ~. ~ : ' :,, .

i3279~3 MSGS(4)' MSG$t5)--"B. CONTINVE -- FIT CUMENT DATA."
MSGS(6)=" "
MSGSt7)='lC. P~T RESULTS ON SCREEN.
MSGS~8)=" "
MSGSt9)--"D. PLOT RESULTS ON PLOTTER."
MSG$(10)=" n McGS(l~ lE. PRINT RESULTS ON PRINTER."
MSGS(12)--" "
MSGS(13)s"F. RETURN TO ~L~IN MENU."
NRET=6:RCS(l)--"A":RCS(2~="B":RCs(3)="c":Rc$(4)a"D":RcS(s)="E~:RCSt6)="F"
CALL MESSAG
IF RETS~:~"F" THEN 1971 NDIN=l:NVIN=l:NLIN=l:NHIN=l:NPIN=l 1971 IF RETS<~"A" THEN 1111 NDIN=l 1111 IF RETS~>"B" THEN 776 FOR I=l TO UVL:VLD(I)=VLP~ VIM(I)=VIR(I):NEXT I
FOR I=l TO NLD:FLD(I)=FIR(I):FIM(I)=FIR(I):NEXT I -FOR I--l TO NHD:HDL(I)=HDR(I):HIM(I)=HIR(I):NEXT I
FOR I=l TO NPO:PRO~I)=PE~R(I):PE`~M(I)=PIR~ NEXT I
JST=101 - ' RMS~r=100000 776 IF RETS~"D" THEN 773 IF NPLOT~>0 THEN 762 BEEP:GOTO 900 762 CLOSE #l:CLOSE #2 FOR I~l TO 6:LPP(I)--l:NEXT I
NAXSs~0 'SEF TO 1 TO INDICATE THAT AXES~ETC HAVE BEEN PLOTTED
909 MSGS(l)'"PLOT THE FOLLOWING ON THE HP PLOTTER:"
MSGS(2)--1' "
MSG$~3)~"A. EXPERIMENTAL PLASMA SPECTRUM."
MSGS(4)--"8. CALCUEATED PLP~SMA SPECTRUM."
MSGS~5)--"C. CALCULATED VLDL COMPONENT SPECTRU~
MSGS~6)--"D. CALCUWI~TED LDL COMPONENT SPECTP~W."
MSGS~7)~"E. CALCULATED HDL COMPONENT SPECTRUM."
MSGS(8)--"F. CAI.CULATED PROTEIN COMPONENT SPECTRUM."
MSG$~9)--"G. PRINT DATA ON PLOT."
MSGS~10)'"X. EXIT."
NRET-8:RCS(l)-"A":RCS(2)~"B":RCS(3)-"C":RCS(4)--"D":RCS(S)--"E":RCS(6)-"F"
RCS(7)-l'XI':RC$(8)-"G'l:CALL MESSAG
IF P~ET$~>"X" THEN 6900 IF NAXS~0 THEN 900 PLOTS-I'LTN+ITS:PRINT #l~PLOTS
PLOTS--PAS+PUS+FNNS~0)+FNNS~0)+PU$+FNNS~0)+FNNS~10)~ITS
PRINT ~ PLOTS
PRINT #l~"SP;n 'STORE PEN
CLOSE #l 6900 IF RET$~>"A" THEN 881 FOR S~51 TO UZP:PLT~ PAT(I):NEXT I
LPP~ 1000 881 IF RETS~'8" THEU 882 FOR I~51 TO NZP:PLT~ PL~I):NEXT I
LPP~2)--1000 ' ' '''~ ' -.' ~
:
- ' . ' .
.
' ' .

~ 1327993 882 IF RETS~>"C" THEN 883 FOR I~51 TO NZP:P1T~I)=C(l)~VLD(I)+c(5)~VIM(I):NEXT I
LPP(3)-1000 '------ REMOVE
' FOR I=l TO KK:PRINT C(I):NEXT I:INPUT JUNX
FOR I-51 TO NZP:PRINT VLD(I),VIM(I~,PLT(I):NEXT I
INPUT JUNK '------------ REMOVE

883 IF RETS<>l~Dl~ T~EN 884 FOR I-51 TO NZP:PLT(I)=C(2)*FLD(I)+C(6)~FIM(I):NEXT I
LPP(4)=1000 884 IF RETS~>"E" THEN 885 FOR I=51 TO NZP:PLT(I)=C(3)~HDL(I)+C(7)~HIM(I):NEXT I
LPP(5)=1000 885 IF RETS~>"F" T~EN 9111 FOR I=51 TO NZP:PLT(I)=C(4~PRO~I)+C(8)*P~ NEXT I
LPP(6)=1000 .: 800 MSGS(1)="SELECT LINE TYPE FOR PLOT:"
MSGS(2)-" "
MSGS(3)="A.
MSGS(4)="B. _ _ _ _ `' MSG5(5)~"C. _ MSGS ( 6 ) =" D . _ . _ . _ . _"
MSGS(7)-"E. _ _ _ _ "
MSGSt8)--"F _ _ _ _ _ _ _ "
: MSGS~9)-"G . . . . ."
NRET~7:RCS(l)-"A":RC$(2)-"B"-RCS(3)-"C":RCS(4)="D"
RCS(5)-"E":RC$(6)'"F":RCS(7)="G":CALL MESSAG
: IF RETS-"A" T~EN LNT-C
IF RETS~"B" THEN LNT-2 IF RET$~"C" THEN LNT-3 IF RETS-"DN THEN LNT~4 IF RETS-"E" THEN LNT-5 ` IF RETS'"F" THEN LNT-6 . IF RET$~"G" THEN LNT'l FOR I-l TO 6 IF LPP(I)~10 THEN 841 LPP(I)~LNT

CLOSE #l:OPEN PCOM$ AS #l PLOT$'INS+ITS+IPS+PlXS+PlYS+P2XS+P2YS+ITS
PLOTS'PLOTS+SC$+XMN5+XMxS+YMNS+YMXS+ITS
PRINT ~l,PLOTS 'INITIALIZE
PLOTS-"PSI'+N10'~+IT$ 'PAPER SIZE - 8.5 X 11 PRINT #l,PLOTS
PLOTS'SP$+FNNS(l)+ITS: PRINT ~l, PLoT$
PLOTS'VSS+''9.5;l':PRINT #l,PLOTS 'SELECT PEN VELOCITY
PLOTS'PAS+PU$+FNN$(0)+FNN$~0)+PUS+FNNS(O)+FNNS(lO)+ITS
PRINT #l,PLOTS
PLOT$-IN$+IT$+IPS+PlXS+PlYS+P2XS+P2Y$+ITS
PLOTS~PLoT$+SC$+XMN$+XMXS+YMNS+Y~SXS+ITS
PRINT #l,PLOTS 'INITIALIZE
PLOT$'"PS"+"10"+IT$ 'PAPER SIZE ' 8.5 X 11 PRINT #l,P~OT$
PLOTTER PARAMETERS
PNA'1 ' PEN NUM~ER FOR AXES
PNL-l 'PEN NUMBER FOR LETTERING
PNS'2 'PEN NUM3ER FOR SPECTRA

..

.
:
. .
;` :

: ~ ` . . ' ~ ' ' '' ': ' `

:

IF NAXS~>0 T~EN 901 REM **~ PLOT AXES ~**~
PLOTS=sPs+FNNS~pNA)+ITs:pRINT #l,PLOTS
PLOTS=VSS+"9.5:":PRINT #l,PLOT$ 'SELECT PEN VELOCITy PLOTS~PA$+PUS+FNNS(O)+FNN$~0)+PD$+FNN$(0)+FNNS(lO~+ITS
PRINT #l,PLoT$
PLOTS-PAS+PUS+FNNS(0)+FNN$(0)+PD$+FNN$(10)+FNNS(0)+IT$
: PRINT ~l,PLOTS
PLOTS5TLS+FNNS(0)+FNNS~.8)+ITS:PRINT #l,PLOTS 'TIC SIZE
PRINT #l,FNPU$(0,0) POR I~l TO 20:P~I/2:PRINT #l,FNPUS(P,0):PRINT #l,XTS:NEXT I
PRINT #l,FNPUS(0,0) FOR I=l TO 20:P=I/2:PRINT #l,FNPUS(0,P):PRINT #l,YTS:NEXT I
PLOTS="SR"+"1.5"+CS+"3.0"+ITS:PRINT #l,PLOTS
WVNS="WAVENUMBERS":LWV=-LEN(WVNS)/2-.1667 PRINT #l,FNPU$(5,-1!) XLBL$=CPS+FNxy$(Lwv~o)+ITs+LBs~wvNs+LT$:pRINT ~l,XLaL$
ITLS="INTENSITY":LIT=-LEN(ITLS)/2-.1667 PRINT #l,FNPUS(-.3,5) YLBL$=CPS+FUXYS(LIT,O)+IT$~LBS+ITLStLTS
YLBL$ = "DI0,1;"+YLBLS:PRINT #l,YL8LS:PRINT #l,"DI;"
PLOTS='~sR~+~1.0~+C$+~2.5'~+ITS:PRINT #l,PLOTS
- YSR=WVR-(5~RINC) YLABS(l)=STRS(YSR):YLABS(l)=RIGHT$(YLAB$(1),LEN(YLABS(l))-l) FOR J-2 TO ll:I=J-l YL-YSR+(I~RINC):YLAB$(J)-STRS(YLI:LY-LEN(YLAB5(J)) YLABS(J)8RIGHTS(yLABs(J)~Ly-l):NExT J
FOR I=0 TO 10:J=ll-I:PRINT #l,FNPU$(I,-.3) LXL-LEN(YLAB$(J)~:LXL--LXL/2:TXL=INT(LXL)-LXL
IF ABS(TXL)~.00001 THEN LXL=LXL+.1667 CHANGE -- NUMBERS AXIS
' IF ABS(TXL)>.00001 THEN LXL-LXL-.1667 XLBLS-CPS+FNXYS(LXL,0)+LBS+YLABs(J)+LTS
' PRINT Yl,XLBLS:NEXT I
`~ 901 REM ~ PLOT SPECTRU~
PLOTS'SPS+FNNS(PNS)+ITS:PRINT #l,PLOTS 'SELECT PEN
PLOTS-VSS+~38.1;":PRlNT #l,PLOTS 'PEN VELOCITY
XMN-0:XMX-10:YMN~0:YMX-10000 XMNS-FNNS(XMN):XMXS'FNN$(xMx):YMNS-FNNS(YMN):YMXS-FNNS~YMX) PLoTs~scs+xMNs+xMxs+yMNs+yMxs+ITs:pRINT ~l,PLOTS
SELECT LINE TYPE
LTPS-"3.0" 'PERCENTAGE OF DIAGONAL ON PLOT FO~ PATTERN
IF ~NT~3 THEN LTPS-"2.0"
IF LNT~l THEN LPTS-"0.5"
PNL-2 'PEN TYPE FOR PLOT
IF LNT-l T~EN PNL'l 'DOTS USE BIG PEN
PLOT$'5PS+FNNS~PNL)+ITS
PRINT tl.PLOTS 'SELECT PEN
LNT$~LT~+STR$~LNT) IF LNT'0 THEN LTPS-""
IF LNT~0 THEN LNT$-"LT"
PLOTS-LNT$+CS+LTPS+ITS:PRINT #l,PLOTS
PX1-S-~WVR-NMT1)/RINC) PYl'(10~PLT~NMTl))/SCLMX
` IF PYl>10 THEN PYl-10 i IF PYl~.001 THEN PYl'.001 ! PY1'1000~PYl PRINT ~l,FNPU$~0,10000) PRINT ~l,FNPUS~PXl,PY1) FOR I-NMTl~l TO NMT2-l:J-I~l 1 ... ... .. ... ... . .... .,. ..... .. . ,... -~r .
"~
' , , : ' . . . .. .
" ': ,, ' ' ' . '' ' ' ' :. ' . ' ' ., ' ~ . . . '. ' : , ' .

~ -- 13~7393 PX=5-((WVR-J)/RINC) PYD(1~PLT(J) )/SCI,MX
IF PY>lO THEN PY=10 IF PY<.OOl THEN PY-.OOl PY=lOOO~PY
PRINT Yl~FNpDs(pxlpy) NEXT I
PX2~5-((WVR-NMEl)/RINC) PY2-(lO~PLT(NMEl))/SCLMX
: IF PY2>10 THEN PY2~10 IF PY2~.001 THEN PY2-.001 PY2-lOOO~PY2 PRINT #l,FNPUS(PX2,PY2) FOR I=NMEl+l TO NME2-l:J=I+l PX=S-((WVR-J)/RINC) PY'(lO*PLT(J))/SCLMX
IF PY>lO THEN PY=10 IF PY<.OOl THEN PY=.OOl PY=lOOO*PY
PRINT ~l,FNPDS(PX~PY) NEXT I
PLOTS="LT"+ITS:PRINT #l,PLOTS
PLOTS-PAS+PUS+FNNS(O)+FNNS(O)+PUS+FNNS(O)+FNNS(lO)+ITS
PRINT #l,PLOTS

9111 XMN=O:XMX=lO:YMN=O:YMX=10 XMN$~FNN$(XMN):XMXS-FNNS(XMX):YMNS=FNNS(YMN):YMXS=FNNS(YMX) PLOTS=SC$+XMNS+XMXS+YMNS+YMXS+ITS:PRINT #l,PLoTS
PNL=l 'PEN TYPE
PLOTS-SPS+FNNS(PNL)+ITS:PRINT ~l,PLOTS ISELECT PEN
PRINT #l,FNPUS(O,O) RSM ~ PLOT DATA ON PLOTTER ~*~
PLOTS-"SR"+ITS:PRINT ~l,PLOTS
PRIUT #l,FUPUS(O,O) PTIT$-FILS
PRINT #l,FNPUS(4.0,12.5) 'LOCATES PEN FOR PRINTING
LBLS'LBS+PTITS+LTS:PRIUT #l,L8LS
PRINT #l,CP$+IT$
PRINT #l,CP$+IT$
PLOTS~LBS+DATES+LTS:PRIUT #l,PLOTS
PRIUT #l,CPS+ITS
PLOT$'L8S+TIMES+LTS:PRINT #l,PLOTS
PRINT #l,CPS+ITS
PRIUT #l,CPS+ITS
PQINT #l~LB$;"RELAT~vE LIPOPROTEIN CONCENTRATIONS:";LTS
PRINT ~l,CPS+ITS
PRIUT #l,CP$+ITS
PRINT #l,CP$+IT$
PRIUT #l,LB$;HVLDL -->";:PRINT #1,VSING "~.~##";ClPR;
; PRIUT ~l,LTS
PRIUT #l,CP$+IT$
PRINT ~l,LB$7~LDL -->";;PRINT fl,USING "~.###~;C2PR;
i PRIUT #l,LT$
PRIUT #l,CP$+ITS
PRINT ~l,LBS;HHDL -->";:PRINT Yl,USING "#.~##";C3PR;
PRINT ~l,LTS
PRIUT ~l,CP$+ITS
PRINT #l,L85;"PRoT --~";:PRIUT ~l,USIUG "#.##~";C4PR:
PRINT #l,LT$
PRIUT ~l,CPS+ITS

'. ' .

:'~ ,, ~ . ' " ' .

13~7993 PRlNT #l~LBS;"~MSD -->";:PRTNT Yl,USlNG ~###.##~;RMS;
PRINT #l,LTS
- ~ PRINT #l,CPS+ITS
PRINT #l,LBS;"TSD -->";:PRINT ~l~USING "###.~#";T~MS;
PRINT #l,LTS
PRINT #l,CPS+ITS
PRINT #l,LB$;"CORR -->";:PRINT #l,USING "#.#~##";CRC;
PRINT #l,LTS
PRINT #I~CPS+I~S
P~OTS-"LT"+ITS:PRINT #l,PLoTS
VJP=8.0 FOR I-l TO 6 IF LPP(I)<O THEN 3773 VJP=VJP-.S
PRINT #l,FNPUS~4.0,VJP) 'LOCATES PEN FOR PRINTING
LNT=LPP ( I) LTPS="3 . O" ' PERCENTAGE OF DIAGONAL ON PLOT FOR PATTERN
IF LNT<3 THEN LTPS=''2~0'~
IF LNT-l THEN LPTS=~o.5 PLOTS=SPS~FNNS~PNL)+ITS
PRINT #l,PLOTS 'SELECT PEN
LNTS-"LT"+STRS~LNT) IF LNT=O THEN LTPS=""
IF LNT-O THEN LNTS="LT~
PLOTS=LNTS+CS+LTPS+IT$:PRINT ~l~PLOTS
PRINT #l,FNPDSt4.7,VJP):PRINT #l,FNPUS(5.0,VJP) PLOTS-L~S+LPQS(I)+LTS:PRINT ~l,PLOTS

PLOTS=PAS+PUS+FNNS(0)~FNNS~O)+PuS~FNNS(O)+FNNS(lO)+ITS
PRINT #l,PLOTS
PNL-2 'PEN TYPE
PRINT #l,"SP;" 'STORE PEN
; GOTO 900 773 IF RETS~"E~ THEN 874 IF NPRNT<>O THEN 766 8SEP:GOT0 900 766 NPSC-l LPRINT:LPRINT:LPRINT:LPRINT:LPRINT
LPRINT BLKS;"NMR LIPOPROTEIN ANALYSIS FOR ";FILS
LPRINT:LPRINT:LPRINT:LPRINT:LPRINT
LPRINT BLXS;"RELATIVE LIPOPROTEIN CONCENTRATIONS:"
LPRINT
LPRINT BLXS;"VLDL -->";:LPRINT USING "#.Y###~;C1PR;
LPRINT U~"t:LPRINT USING "#. #";SE(l)::LPRINT "~"
LPRINT BLKS:"LDL -->~::LPRINT USING "Y. #":C2PR;
LPRINT "~";:LPRINT USING "#.Y";SE(2);:LPRINT ")"
LPRINT BLXS;nHDL -->";:LPRINT USINC "#.#~###":C3PR:
LPRINT "t"::LPRINT USING "#.###~#":SE(3)::LPRINT ~
LPRINT BLK$:~PROT -->";:LPRINT USING "#.###Y#'~;C4PR;
LPRINT "~";:LPRINT USING "#.##yy~";SE(4);:LPRINT ")"
LPRINT
LPRINT
LPRINT 8LRS;''ROOT MEAN SQUARE DEVIATIONS ~ CORRELATION COEFFICIENTS:"
LPRINT
LPRINT BLXS:~RMSD FOR TOTAL METHYL ~ METHYLENE FIT -->";
LPRINT USING n~##.#~n;TRMS
IF NREG~ THEN 765 IF NREG-l THEN RGNS'"RMSD FOR METHYL REGION (FIT) ~~>~
IF NREG~2 THEN RGN$~"RMSD FOR METHYLENE REGION ~FIT) ~~>"
LPRINT BLX$:RGNS :LPRINS USING "~#~.~#~"~RMS

~, , , , '' ', :' ~ . . ' ~ . :

13279~3 .
.
765 RGNS="CORRELATION COEFFICIENT ~~>"
LPRINT sr~s, RGNS; LPRINT US I NG '~#~Y##Y'~:CRC
LPRINT
~ LPRINT
- DLTA=JST-51 NNEW=NSTOR-DLTA
LPRINT BLRS;"INITIAL STARTING DATA POINT FOR PLAS~A -->";NSTOR
LPRINT BLK$;"5TARTING DATA POINT FOR BEST LEAST SQUARES FIT -->";NNEW
LPRINT
LPRINT
- LPRINT ~LKS;"OPTIONS SELECTED ~~>"
LPRINT
IF NREG~l THEN LPRINT ~LKS;"METHYL REGION (ONLY) FIT~"
IF NREG-2 THEN LPRINT BLKS;"MET~YLENE REGION (ONLY) FIT~"
IF NREG=3 THEN LPRINT BLXS; "METHYL AND METHYLENE REGIONS FIT. "
LPRINT
~LNS="PHASE cORRECTION -->"
LPRINT
LPRINT BLK$;8LN$;
THQ-THT
LPRINT USING "###.##";THQ;:LPRINT " DEGREES"
LPRINT
7773 IP NCN=0 THEN 7772 LPRINT
LPRINT BLRS;"CONSTRAINTS ~~>"
LPRINT
FOR I - 1 TO K~
I F JCS ( I ) - O THEN 7369 IF I~>l THEN 7301 LPRINT i3LXS;"VLDL COMPONENT CONSTRAINED TO "; LPRINT USING "~ ~";ClPR

7301 IF I~>2 THEN 7302 LPRINT BLKS;"LDL COMPONENT CONSTRAINED TO ";: LPRINT USING ''#~ #U;C2PR
7302 IF ~>3 THEN 7303 LPRINT BLKS;"HDL COMPONENT CONSTRAINED TO "; LPRINT USING "#.#~###";C3PR
7303 IF I~>4 THEN 7369 LPRINT BLKS;"PROTEIN COMPONENT CONSTRAINED TO ";
LPRINT USING "Y~#Y###";C4PR

7772 TSPPS~BLKS+"TSP PEAX INTEGRATED FOR '~FILs LPRINT TSPPS
LPRINT
LPRINT BLKS; LPRINT "INTEGRAL ~~> "; LPRINT USING "~ ###";ARPQ
LPRINT
TSPP5-3LXS~ToP OF PEAK LOCATED AT"+STRS(NSTR+NTQP) LPRINT TSPPS
TSPPS-BLK$+"PEAK INTEGRATED FROM"+STRS(NSTR+IPQl)+" TO"+STRS(NSTR+IPQ2) LPRINT TSPPS
LPRINT BLKS; LPRINT "INTEGRAL NORM~LIZATION FACTOR ~~> ";
LPRINT USING "#~#.##";RNQRM
LPRINT
i LPRINT aLXS;"NORMALIZED LIPOPROTEIN CONCENTRATIONS "
LPRINT
ClPZ-ClPR~RNQRM
CZ PZ'C2 PR~NQRM
C3 PZ - C3 PR~RNQRM
C4PZ-C4PR~RNQRM
LPRINT BLXS ~ ~VLDL --~";:LPRINT USING N #, y Y Y ~ # ~; ClPZ
`~ LPRINT BLXS;"LDL ~~>" LPRINT USING '~#.t~Y~'':C2PZ
LPRINT BLKSt"HDL -->"~:LPRINT USING "#~ ";C3PZ
LPRINT BLKS;"PROT ~~~"; LPRINT USING "~#l#~#";C4PZ

,, ~ ~ . ... .. ... . .... - .. . . ..
. .
~, .

.

.

LPRINT CHRS(12) ~78 END
~*~*~ *~ MATRIX INVEaSION ~*~f ~t~ b~*~*~***
SU8 INVERTlIERR,NCM) STATIC
COPY AM INTO LINEAR ARRAY A
X-0:N-NDM:IERR-l FOR I~l TO N:FOR J~l TO N:K=K+l:AtR)=AM(J~ NEXT J:NEXT I
SEARCH FOR LARGEST ELEMENT
D=1.0:NK--N:
FOR X-l TO N
NX=NK+N:L(K)=K:M(K)=K:KX=NK+K;8IGA=A(XX) FOR J=K TO N
IZ=N*(J-l) FOR I=K TO N
IJ=IZ+I
TST=ABS(BIGA)-A~StA(IJ)) IF TST>=0. THEN 20 BIGA=A(IJ):L(K)=I:M(Kj=J
NEXT I
NEXT J
INTERCHANGE ROWS
I
J-L(K):JTST=J-K
IF JTST~=0 THEN 35 XI~X-N
FOR I-l TO N
KI-KI+N:HOLD -A(KI);JI=XI-X+J:AtXI)-A~JI)~A(JI)sHOLD

. ' INTERCaANGS COLUMNS
I~M(X):JTST-I-X
IF JTST~0 THEN 45 JP-N~
FOR J-l TO N
JK~NK+J:JI~Jp+J:HoLD~-A(JK):A(JK)=A(JI):A(JI)~HoLD
45 IF A8S(8ICA)>0~0000001 THEN 48 SIUGULAR MATRIX ~~> EXIT
~ D~0~:DETaD:IERR~0 : EXIT SU8 . ~
~ DIVIDE COLUMN 8Y MINUS PIVOT (PIVOT IN 8IGA) 48 FOR I-l TO N
IF I~X THEN 55 IX~NX+I
A ( IK) - A(IX)/1-8IGA) ; 55 NEXT I

REDUCE MATRIX

FOR I~l TO N

.
' , ' . ' ~ '~ " ' , ' ;
.

, ~327993 . .

; IK-NK+I:HOLD=~(IK):IJ=I-N
- FOR J=l TO N
~ IJ~IJ+N

IF J~K THEN 65 KJ IJ-I+K
A(IJ)'HOLD~A(XJ)~A(IJ) NEX~ J
NEXT I
DIVIDE ROW BY PIVOT
KJ=K-N
~ FOR J-l ~O N
XJ=KJ+N

A(KJ)=A(KJ)/~IGA
NEXT J
PRODUCT OF PIVOTS
. D-D*BIGA

A(XX)~1.0/BIGA
NEXT K
FINAL ROW & COLUMN INTERCHANGE
X-N
100 X~X-l IF K<'0 THEN 150 I'L(K) . $F I~'K TYEN 120 : JQ-N~(X~ JR-N~( r-FOR J-l TO N
JX-JQ+J:HOLD'A~JX):JI~JR+J:A~JX)'-A(JI):A~JI)'HOLD
120 J'M(X) IF J<'X T~EN 100 KI'K-N
FOR I-l TO N
XI-KI+N:HOLD~A(KI):JI-XI-X+J:AIXI)~-A(JI):A(JI)-HOLD
:I GOTO 100 - ' 1/1AM~ NOW STORED IN A --> COPY INTO AI

FOR I'l TO N:FOR J~l TO N:K~X+l:AI(J,I)~A(X):NEXT J:NEXT I
END SUL
______________________ _______________.______________________ ! SUD MESSAG STATIC
- N2o'LEN~Mscs(2o))/2:N2o~4o-cINT(N2o) LocATE 2,N20,0:PRINT MSGS(20):
SQS~CHRS~205) LOCATE S,1,0:PRINT ~ "i:FOR I'l TO 74:PRINT SQ$;:NEXT I
LOCATE C,1,0:PRINT " "i:FOR I~l TO 74:PRINT SQS~:NEXT I
JCT~l:LM~0:FOR I-l TO 20:LMS~LEN~MSGS~

.. ... " ` ~ ~

-.. , ,~ . , , IF LMS>LM THEN LM=LMS
IF LMS~.l THEN 91 - NEXT I
91 NMS=I-l:LC=CINT(40-(LM/2)) FOR I=l TO NMS:LL=I+7 LOCATE LL,LC,0:PRINT MSGS(I);
NEXT I
IF NRET>.l THEN 92 LLl~LL+2:LL2-LLl+l:LL3=LL2+2:LL4=LL3+2 LOCATE LLl,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQS::NEXT I
LOCATE LL2,1,0:PRINT " "::FOR I=l TO 74:PRINT SQS;:NEXT I
IF NLAG >.1 THEN 93 LOCATE LL3,27,0:PRINT "PRESS ANY XEY TO CONTINUE";
94 RETS=INXEY$:IF RETS="" THEN 94 I P LEN(RETS)>l THEN 95 IF RETS="S" THEN 95 IF RETS<>ECO$ THEN 95 STOP
92 LL=LL+2:LLl=LL+2:LL2=LLl+l:LL3=LL2+2:LL4=LL3+2 LOCATE LLl,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQS;:NEXT I
LOCATE LL2,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQ$;:NEXT I
MGS-''ENTER CHOICE - > ":LOCATE LL,31,0:PRINT MGS;
96 LOCATE LL,49,1:RET$=INXEYS:IF RE~S="" T~EN 96 IF LEN~RETS)>l THEN 911 IF RETS="S" THEN 95 LC-ASC(RETS~
IF RETS<>ECo$ THEN 910 STOP
910 IF LC>96 THEN RETS=C~RS(LC-32) PRINT RETS;:LOCATE 1,1,0 FOR I=l TO NRET:IF RETS-RCS(I) THEN 97 NEXT I
911 BEEP:BEEP:8EEP:FOR I-l TO 5:FOR J-l TO 15 LOCATE LL,55,0:PRINT "WHAT?n:NEXT J
FOR J'l TO 10:LOCATE LL,55,0:PRINT ~' N NEXT J
NEXT I:GOTO 96 97 FOR I~l TO 500:NEXT I:GOTO 95 93 FOR I-l TO NLAG:NEXT I
FOR I~l TO 18:MSGS~I)~"":NEXT I
FOR I~l TO 20:RCS(I)-"":NEXT I:NRET=0:NLAG=0 DUM-FRE(AS) CLS:LOCATE 1,1,0 END SUB
______________________________________________________________________________ SUB MESAG STATIC
CLS:SQ$-CHRS(223) U20'LEU~MSGS~20) )/2:N20D40-CINT(N20) LOCATE 2,N20,0:PRINT MSGS(20) LOCATE 5,1,0:PRINT " ";:FOR I-l TO 74:PRINT SQS;:NEXT I
: JCT~l:LM~0:FOR I'l TO 20:LMS-LEN(MSGS(I)) IF LMS>LM THEN LM-LMS
IP LMS~.l THEN 377 NEXT I
377 WS'I-l:LC'CINT(40-(LM/2~) FOR I'l TO WS:LL-I+7 UOEcxTTE LL,LC,0:PRINT MSGS(I) LLl~LL+2:LL2'LLl+l:LL3~LL2+2:LL4~LL3+2 LOCATE LL2,1,0:PRINT 1~ ";:FOR I-l TO 74:PRINT SQ$;:NEXT I
FOR I~l TO 18:MSG$(I)-"":NEXT I

~, ~. .
.

LOCATE 1,1,0 -DUM=FRE(AS) END SUB
____________________________________________ ____ __ __ _____________ SUB SETIN(INPR) STATIC
IF INPR~.5 THEN 555 MSGS(l)=" PRINT OPTIONS:"
MSG$(2)=" ":MSGS(4)=" "
MSGS(3)-"S. PRINT ON SCREEN ONLY.
MSGS(5)="P. PRINT ON SCREEN AND PRINTER.
NRET=2:RCS(l)-"S":RCS(2)="P"
CALL MESSAG
IF RET$="S"THEN 555 NPT=l MSGS~l)="BE CERTAIN PRINTER IS READY AND PAPER IS ALIGNED!"

555 CLS:SQS=CHR$(219) LOCATE 5,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQ$;:NEXT I
LOCATE 6,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQ$;:NEXT I
LOCATE 14,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQS;:NEXT I
LOCATE 15,1,0:PRINT " ";:FOR I=l TO 74:PRINT SQS;:NEXT I

______________________________________________________________________________ SU8 INPT(MSINS,LSP) STATIC
LOCATE 9,1,0:PRINT SPC~79) LOCATE ll,l,O:PRINT SPC~79) LOCATE 9,~SP,O:PRINT MSINS;
DUM~FRE(A$) END SUB

Claims (12)

1. A method for measuring the lipoprotein constituents of blood, the steps comprising:

storing the NMR spectrum of each lipoprotein constituent as a reference spectrum for that constituent;

acquiring the NMR spectrum of a plasma or serum sample of the blood to be analyzed;

producing a calculated lineshape by adding together the reference spectrum for each constituent in amounts determined by respective constituent coefficients;

adjusting the constituent coefficients to fit the calculated lineshape to the NMR spectrum of the sample; and calculating the concentration of at least one lipoprotein constituent as a function of the value of its constituent coefficient.

2. The method as recited in claim 1 in which the lipoprotein constituents are VLDL, LDL, HDL, and proteins.

3. The method as recited in claim 1 in which the NMR
spectrum includes at least one of the peaks produced by methylene and methyl protons.

4. The method as recited in claim 1 in which the NMR
spectra are chemical shift spectra and the sample NMR spectrum is acquired by:
a) acquiring the NMR signal produced by the sample in an NMR spectrometer; and b) performing a Fourier transformation on the acquired NMR signal.

5. The method as recited in claim 4 in which the NMR
spectrum of each lipoprotein constituent is produced by:
a) separating the lipoprotein constituent from a sample of blood;
b) acquiring the NMR signal produced by the separated lipoprotein constituent in an NMR spectrometer; and c) performing a Fourier transformation on the acquired NMR signal to produce the NMR spectrum of the lipoprotein constituent.

6. The method as recited in claim 4 which includes:
d) shifting the sample spectrum to align a known peak therein with its known chemical shift value.

7. The method as recited in claim-1 in which the calculated lineshape is fit to the NMR spectra of the sample by minimizing the root mean square error.

8. The method as recited in claim 1 which includes storing the NMR spectrum of a non-lipoprotein constituent as a reference spectrum for that constituent and including the reference spectrum for the non-lipoprotein constituent in the calculated lineshape.

9. The method as recited in claim 1 which includes the step of generating a printed report which indicates the concentration of each calculated lipoprotein constituent.

10. Apparatus for measuring a plurality of lipoprotein constituents of blood comprising:
means for storing the NMR spectrum of each one of said plurality of lipoprotein constituents as a reference spectrum for that constituent;
means for acquiring the NMR-spectrum of a plasma or serum sample of the blood to be analyzed;
means for producing a calculated lineshape by adding together the reference spectrum for each constituent in amounts determined by respective constituent coefficients;
means for adjusting the constituent coefficients to fit the calculated lineshape to the NMR spectrum of the sample; and means for calculating the concentration of at least one of said plurality of lipoprotein constituents as a function of the value of its constituent coefficient.

11. The apparatus in claim 10 in which the NMR spectra are chemical shift spectra and the sample NMR spectrum is acquired by:
a) acquiring the NMR signal produced by the sample in an NMR spectrometer; and b) performing a Fourier transformation on the acquired NMR signal.

12. The apparatus in claim 10 which includes printer means for producing a report which indicates the concentration of each calculated lipoprotein constituent.