CA1163908A

CA1163908A - Method for eliminating turbidity in a biological fluid and reagent therefor

Info

Publication number: CA1163908A
Application number: CA000380441A
Authority: CA
Inventors: Shyun-Long Yun; Luis P. Leon; Syed I. Ahmad
Original assignee: Technicon Instruments Corp
Current assignee: Bayer Corp
Priority date: 1980-10-01
Filing date: 1981-06-23
Publication date: 1984-03-20
Also published as: AU7287681A; CH657919A5; AU543239B2; JPH0474000B2; FR2495184A1; IT8168070A0; NL8104304A; IT1144750B; GB2084726A; DE3138602A1; SE8105737L; JPS5780560A; DE3138602C2; SE449005B; GB2084726B; JPH01304898A; FR2495184B1; BE890479A; JPH0151782B2

Abstract

Abstract of the Disclosure A method for eliminating turbidity in a biological fluid by combining the fluid with a surfactant of the general formula:

Description

~ ~390~, . , , . ~
.

.

~ .
.

" .
Il . .
12 ; Backqround of the Invention 13 ! This invention relates generally to a method for effectively ~ clearing turbidity in a biological sample such as human 15 jl serum and to a reagent used for that purpose. More particularly, ' 16 Ij it relates to a reagent comprised of a surfactant and an ~7 Il, enzyme and to its application as a turbidity-removing agent.
! ., lS ,j Turbidity in a biological sample can cause severe problems.

It results in poor or incorrect readings and therefore highly 1~ questior.able determinations.
211l '

2~ ¦¦ Turbidity in serum and plasma samples is usually a ~ 1' serious problem in clinical photometric analysis. It gives 24 1I false data and often yields misleading photometric ~5~! `
'6 ~ . .

-..................... . . .

. ` ' , ' `' ~ ' I .
`

1 1 16390~
708-017 il I ~
I
l . ', 1 I determinatlons of serum lngredlents. The turbldLty ls 2 , believed caused primarlly by the elevatlon of triglycerldes

3 j in serum of patlents havlng hyperllpopro~eineola with or

4 without elevated total cholesterol. Abnormal elevation of

5 ¦ cholesterol ln 3erum has been shown to correlate wlth a 1

6 ! high rlsk artherosclerosls. Determlnation of cholesterol

7 1 and triglycerltes ls lmportant slnce accurate data will help

8 ¦ the doctor to dla~nose patlents wlth hyperLipoprotelnmla

9 ¦ Dnd to predlct certaln heart dlsen3e. Other te~ti such as

10 1 aspartate amlnotran3ferase (GOT), alanlne amlnotransferase j tCPT) and lactate dehydrogenaae tLDII), etc. have ulso suffered 12 1 from the same dlfflcultles as choles~erol or trlglycerlde 13 j determLnation, as mentloned above, when turbld samples are 14 examinet.

6 1 Cllnical eests of turbid serum has been handlet 17 1 by treatl~g samples wlth hlgh concentratlon of surfactants 18 ! such as polyoxyethylated lauric acid (U.S. Patent No~.

19 ¦ 3,853,465 and 4,184,ô48~. Slnce only surfactants were uset, 20 ¦ a rather hlgh concentratlon of surfactant was needed for 21 ¦ an effective clearlng. High concentratlon of surfactants 22 ¦ are often lnteractet wlth other chemlcal or en~yme reactlons 23 ¦ ant causes compllcatlon of analysLs.

24 l 25 ¦ In the present lnventlon, a relatlve low concentra-26 ¦ tlon of surfactant ls used. Thls Ls accompllshed by the 27 ¦ actlon of enzyme tcholesterol esterase or llpase) added to 28 ¦ the clearlng reagent.

29 I .

30 ¦ ~ The exact mechsnlsm of clearlDg by this inventlon _I -2-.. . .

1 1639D ~ I
'08-017 1 !
1 I is not yee known. It is possible to speculate however tha~, 2 ' in cases where patlencs have hyperlipemia, the turbidity 3 ! found ln serum samples ls due primarlly eo an elevated trl-4 ! glycerlde content.

5 ~
6 ¦ Triglycerldes are water-lnsoluble, and usually 7 1~ buried lnslde the fat core wlth cholesterol eseers ln 8 1 lipoprotein complex. The clearing of a llpemlc sample mu3e 9 ¦ be brought aboue by the dlsrupeure of lipoproeeln by a lO ¦ surfaceane such as laurlc acld dleehanolamide (DEA), followed ll ¦ by the hydrolysis of tri~lycerldes by the enzyme base. The 12 1 surfactant also aids the dlssolutlon of fatey aclds released 13 ¦ eherein. Wlthout enzyme, there is no clearing.

Ie is the ob~ect of ehls inventlon to provlte a 16 i rea~ent which is effective ln clearln~ turbldity in biolo~ical 17 1 samples and to a method of effecelng same, parelcularly ln lô ¦ cases where ehe sample 19 eo be photomeerlcally assayed or l9 analyzed for a particular component such as cholesterol.
201 .

211 Summary of the Invention 22 ¦ In accordance wlth thls lnventlon, there ls claimed 23 ¦ a reagent effectlve in clearlng eurbldley of a blological 241 sample comprlsing a surfactant of the formula:

26 I < (CN2CN20) 27 ¦ (CH2CN20) 29whereln R is alkyl or alkenyl containing from 5 to 17 carbon `atom3, x and y are each 1; and an enzyme ~elected fro~

~1 _3_ .... .. . .. , ~

~ 017 li Il ~ 163 2 ¦ cho estero1 esteras~ or lipase or ~ixtures thereof.
3 Preferably~ the above reagent in aqueous buffered form 4 will comprise ~rom about 0.05 g~dl to about 2.5 g/dl of surfactant, and preferably from O.l g/dl to about 0.5 g/dl, and at least about 0.025 U/ml of enzyme (cholesterol esterase 6 based on the total formula;ion. The resulting formulation will have a pH in the range from about 5.5 to about 7Ø

9 When the formulation contains a lipase enzyme, the surfactant comprises from about 0.05 g/dl to about 2.5 g/dl, ! and preferably from O.l g/dl to about 0.5 g/dl, and said i enzyme comprises at least about l.0 U/ml of the resulting 1~ 1, formulation, said formulation having a pH in the range fro~
13 I about 5.5 to about 8Ø
I
In both enzyme formulations, the buffer employed can 16 be a maleate, in the form of the sodium or potassium salt;
l7 ! a phosphate; a borate; a citrate; a succinate; an imidazole-l~ ~ acetate buffer; Tris; etc. Any suitable buffer san be usedhowever. Such buffer i5 any one which will maintain a constant 19 ¦ pa in the desired range without interfering with any of 1o the components.
211 . , ~ , 22 ¦ When a maleate buffer is used, e.g. the potassium or _3 ¦ sodium salt, it is added in amounts to provide from about 2~ ¦ 0.05 M to about 0.5 M and the pH of the resulting formulation is from about 5.0 to about 7Ø I
'5 . - , .

21 Similar concentrations are used for the other buffers.
_4_ .
, ~ _ . . . ~., ~

708-Ol/ I
jj 116390~
Il . , `'- .
I In addition to the above components, a solubility enhaneer . can be included in the above enzyme formulations. Sueh 3 enhancer comprises any material which aids the suraetant 4 in solubilization. For example, bile salts are particularly 6 effective such as sodium cholate, sodium deoxycholate, ete.
~ In another preferred embodiment the surfaetant is of ¦ the above formula in which R is alkyl such as lauric aeid g 1 diethanolamide or oleic acid diethanolamide.

10 ¦ The enzyme is derived from animal, e.g. animal pancreatic

11 tissue or mierobial souree.
I . i 1~ l!
j, Biological samples suitably treated include human sarum ¦ and plasma.

15 jj The reagent described above whan combined with a biological 16 ¦I sample results in an effeetive turbidity-elearing effeet.
17 !I The reagent is commonly provided in aqueous buffered form.
Ig I
19 I Also withln the purview of this invention is a reagent, effective in clearing turbidity of a biologieal sample to be photometrically assayed or analyzed which eomprises ,1 1 at least one surfactant of the formula: j '~71 ~ ¦ ~ CH2CH20)XH
2~ 1 ~- -N
'5 ¦ ~CH2CH20~ y~

27 wherein R is alkyl or alkenyl oontaining Erom 5 to 17 caFbon ~ ~
-5- .' r.~q ll~ ' ~_~ .

7~8-0l7 116390B
, i .
atoms, x and y are whole number int er w ose sum is no 3 e ~ec~
. greater than ll; and an en~yme ~se~e~ from cholesterol 3 ¦ esterase or lipase or mixtures thereof.

4 A preferred reagent is one having a surfactant of the S above formula in which R is alkyl, preferably lauryl, and 6 the sum of x and y is 5 and the enzyme is a lipase. Formula- !
7 tions with this reagent desirably include polyethylene glycol 3 1 p-isooctyl phenyl ether or other suitable surfactants.
9 I , .
10 ¦ Another preferred reagent which will produce effective 1I clearing of turbid samples in the p~ range of 2 to lO is ¦1 a mixture of two surfactants, namely, lauric acid diethanolamide I ¦l (x = y = l) and epoxylated lauric acid ~x + y = 5).

1~ ¦I Enzyme formulations as previously described àre prepared 15 1 using the surfactant shown above.

17 Suitable surfactants embraced by the above formula 18 ¦ include lauric acid diethanolamide, myristic acid diethanol-19 ! amide, capric acid diethanolamide, oleic acid diethanolamide and coco acid diethanolamide.

1 This reagent when combined with a biological sample ~, effectively clears turbidity in the sample and allows it to be accurately photometrically assayed or analyzed. In 24 this manner, a sample to be photometrically analy2ed for 2S cholesterol is beneicially treated.
26 - -6- ! ~

.,... . ' . ..

~: ~

1 1! ~ 3 ~

1 Detailed Descrlption of ehe Inveneion 2 I The present invention, in one aspact, involves a 3 reagent and method which effectively clear turbidlty of a 4 i biological sample by the employment of a particular surfac-5 I tant and enzyme. ln this aspect, the surfactant has the 6 forrula:

- ~ (CH2CH70) H
8 ~-C-N <
9 (CII~CH70) H

wh~r~in R i~ alkyl or alkonyl of from S to 17 carbon atoms,

12 ~ and y ar~ ~ach 1 Pref~rably~ R i~ al~yl exemplifled by /~ ~,e7'~o~0 ~
_~ 13 , lauric acid d~ ~r~ti~.

15 I The enzyme component is cholesterol esterase or 16 lipase or mi~tures of the two.

18 The resulting formulaeion has been found, quite 9 ' iurprislngly, to be lilghly effeccive in clearing turbidity in a biological sample. It is therefore enormously useful 21 in converting a biological specimen which is curbld to one 22 which i~ clear;
23 ;
24 The sample so treated can be colorlmetrically 25 , assayed or analyzed for any particular component so long as 26 j the reagents employed in the assay do noc inhlbit or lnter-27 I fere with the interactlon between surfactant anù enzyme and .
28 ~ vice-versa.

29 , .. ' ~ ~

! I , .

708 0~ 1 6 3 9 ~ ~ 1 I 1¦ Typical assays that can be carried out utilizing the ¦ above reagent include cholesterol, triglycerides and creatine ; ! phosphate kinase determinations.

5 I The enzyme component can be derived from an animal i -source such as pancreatic tissue or from microbial source.

7 In a second aspect of thls invention, a reagent and ~ method are taught which effectively clear a turbid biological 9 ¦ sample which is to be photometrically assayed. The reagent lO ¦ includes at least one surfactant, more broadly defined than Il " above, having the formula:
i' 1

13 i ,l!i /(CH2CH20) x13 '~ . R--C-N~
\(CH2CH20) yH
'51i -- . I
16 I wherein R is alkyl or alkenyl containing from 5 to 17 carbon m4~, x and y are whole number integers whose sum is no li greater than 11; and an enzyme selected from cholesterol 19 esterase or lipase or mixtures thereof.

In a preferred embodiment, the surfactant is as above 21 in which R is alkyl and x and y are each 1. Examples of 22 the latter include lauric acid diethanolamide, myristic acid diethanolamide and capric acid diethanolamide. Also 2~ ¦ preferred are surfactants in which R is alkenyl and x and .~ y are 1 such as oleic acid diethanolamide and coco acid 2~ diethanolamide. In another preferred embodiment, the surfactant ,, is as above in which R is alkyl and the sum of x and y is 27 5.

,~ ,` ~=~ ~

... .. ..

1 î63~0~
. .
.

1 The enzyme componen~ can be deriYed from an anlmal 2 source such as pancrea~ic ~issue or from a microbl;ll source.

4 When ~he above reaBent is employed for assaying purposes, i~ is combined in aqueous buffered form with a bio-6 logical sample. The turbid sample is clenred and is re~dy 7 for ~he ~ssny.

9 Sample~ which are efEiciently cle3red and prepared ; for 3ss3y Include human s~rum and pl3sma.
11 ' 12 The me~hod and reagent of this inven~ion nre unique 13 ' in at l~agt two respects. They permi~ the de~ermlnntlon of

14 components in biological samples in a clear free state with-out ~urbidi~y in~erferences and, in additlon, because of che 16 inceraction between the partlcular surfac~an~ and enzyme em-17 ployed, permlt one to use lesser amounts of en7yme than are 18 , normally used as in the case of cholesterol determination.
19 ! In this latCer regard, ~he employment of smaller amounts with-~eS' 20 i ouc lessening in r~d~ of reaction can be viewed as an im-21 I provement ln the rate of enzymaeic reac~ion, 22 ;
23 I The mo~t common clinical determination of choles-~
24 terol in a biological fluid is the to~al cholesterol which includes both free cholesterol and cholesterol esters. ~oth 26 I cholesterol and its esters are present in serum wlth other 27 1 lipids and various proteins ln mlcromolecular complexes called 28 i; lipoproteins and cholesterol esters normally exist as a ~a~or 29 ~ component (60-80X~ of the total cholesterol. They are generally water lnsoluble and are normally burled lnside il _ g _ 1 ~639~fl :'.

.1 1 , the complex and inaccessible to enzymes. In ehe detcrmina-2 I tion of to~al cholesterol by a wholly enzymic meehod, 3 whe~her aueomated or manual, boeh cholesterol and choles-4 ~ terol esters must first be liberated by a suitable surfac-' tant. Cholesterol esters are then hydrolyzed by cholesterol 6 Ij esterase to yield froe cholestcrol which i9, in turn, 7 oxidized by cholesterol oxidase to form choles~enone and 8 . hydrog~n peroxide.
1~ Tll~ hur~in di.~los~d m-thod cun h~ a~ d In 11 ;, automat~d fa~hlon as by tilC cmploym~nt of an automatlc 12 analyzer or it can bc done mnnually.
13 :!
14 , In preparing the formulations eor use in assay-lS ' ing-by the method of thi~ inventlon, an aqueous solution lt9 ~ i~ formulated which contains, in addi~ion to the surfactant 17 ' and enzymd, o~her reference materials which are known in 18 ' the art and are utilized for such purpose.
19 1 ' 20 '! For ex~mple, in cholesterol assaying, the follow-21 1 ing components arc employed in the ranges shown hcrein-22 ¦ below~

23 ~, 24 ¦ Componen~ Cholesterol Assay 25 l Peroxidase 0.8 - 7.0 U/l 26 ¦ Cholesterol oxidase 0.025 - 0.3 U/ml 27 ~` Cholesterol esterase .025 - .3 U/ml 28 1¦ Surfactant .05 - .5 g/dl I .

29 ¦ Sodium cholate 0.05 - 0.5 g/dl Sodlum p-Hydroxybenzo2te 2.5 - 6 g/dl 'I ~ .
-10- . ,.

~ 16~0~

., .
1 i Component (con~inued) Cholesterol Assay 2 j 4-Aminoancipyrine 0.5 - 7 . 0 mM
3 ¦ Maleic acld O.l - 0.5 M
4 I pH 5.5 - 7.0 S ! Sample/Reagent r ItiO 100 - 400 7 '¦ In the above Eormulaeion, i~ is preferred to use 8 ' choleseerol es~erase Eron an aninal source, a.B. pancreas;
9 ¦ however, equivalen~ re~ults are ob~ained with cho1escerol es~erase from a microbial source.
11'1 .
2 ;I EXAMPLE I
.j :
13 Clearin~ as a function oE cholesterol e~erase ! I i 14 ~I Three nl of clearlng reagene whlch contains ~ potassium-maleate (,0.1 `I)~ sodiu,m cholate (0.25 g¦dl), 'I ~ ~-7o~,~2 .
16 l laurlc acid ~ r~ i~e (0.2 g/dl), cholesterol esterasé

17 j (0.08 U/ml to a.s Ulml) flnal pH 6.0 ls mixed wlth 0.025 ml 18 l of lipemic seru~ (trlglycerides or aboue 1400 mg/dl). The 19 reaction mlxture turn's clear, within 10 mlnutes ae 45C.

20 ~ The cholesterol esterase for clearing can be from pancreas 21 or microorganisns.

22'.1 1 . .
23 EXA~PLE lI
. . _ ' 24 Use Eor cholesterol determlnatlon ., I
25 ~¦ For end polnt chemistry as lllustrated in thls 26 l example the lngredients for cholesterol assay are thus s7 1l included lnto the clearing reagent.
2~ i1 1 . ;
29 ,¦ Three ml of a formulatlon which coneains choles-eerol eseerase (0.125 Ulnl), choleseerol oxidase (0.125 Ulml), !
! ~
.. I -11-' I

, 1 ~6390~
708-017 ,1 .i , .
'I
1 peroxidase (1.6 U/ml), 4-aminoantipyrina (0.6 mM), sodium 2 I hydroxyben7Oate (25 mM), sndium cholate (0.25 g/dl), laurlc 3 acid diethanolamide (0.2 g/dl) and potasslum maleate (O.l M), 4 pH 6Ø This whole reagent ls mlxad with O.OZ5 ml of lipemic 5 ,I serum sample. The mlxture is then incubaead a~ 45C for 6 ' 4-5 minutes. Th~ total cholesterol is then determlncd by 7 m~asuring tha color intansity at 5'0 nm. Without the clearlng 8 eEEect of lauric acid diethanolnmidc nnd cholesc.rol estcra~a, 9 ll the detcrmlnntion of chol~st~rol in turbid samplas always 0 j gives erroncous results.
11;
12 t EY.~MPLE II r 13 Clearin~ by candida lipnse (candida cylindracea) 14 ~I Three ml of a clearing reagen~ containing lauric

15 ¦ acid diathanolamLde (0.2 g/dl), Na cholate (0.25 ~/dl) and

16 j lipase ('5 U/ml) and maleate buffer (O.l M), pH 6.0, are

17 j mixed with 0.025 ml lipemic serum. The turbld sample will

18 I turn clear afcer 5 minutes incubation at 45C

19 !

20 1 When an equivalent amount of clearing agene com-

21 prising a mixrure of lauric acid die~hanolamide and apoxyla~ed

22 ' lauric acid tx + y - 5) is used, compar~ble clearing results.

23 '

24 E.Y-~MPLE IV

25 i Three ml of a clenring reagent containing a surfac-

26 ll tant of the formula:
27ll ~ (CH2CH20) H .

29 j (CH2CH20) H

i -12- .
, .

il 1~639 708-01~ `!
;, ' in which x + y - 5 (0.2 gldl), Triton ~-l00 (0.4 g/dl), 2 , po~assium maleate (0.2 M) and lipase (25 U/ml), pH 6.0, ls 3 ~ mixed with 0.05 ~1 lipemic sample nnd incubated at 45~C.
4 ;! The turbid sample turni clear after 3 minutes.
5 '!
6 1 ~ The rate of clearing is enhanced by increasing 7 I the buEfer concentratlon.
8 'I
9 '¦ Similar r~sults are obtaln~d by using higher 10 ,~ concentratlons of epoxylated lauric acld (x + y ~ 5) wlthou~ j the assls~ance of Trlton ~-100 3 ! E~A~PIE V
-I
4 ;l A. Formulation 5 1 ~3 dlagnostic reagent formulatlon ls prepared as a 6 1 one liter aqueous solutlon usln~ the following ingredients.
17 i 8 ~¦ Ingredient Concentration ,¦ Malic Acid 11.6 g 20 'I ROH 10.0 8 21 !I EDTA (R2) Z-7 22 ! Na Cholate 5.8 mM
23 ~¦ Na p-Hydroxybenzoate 25.0 mM
24 ~I 4-Aminoantipyrlne 0.6 mM
25 I Lauric Acid Dlethanolamide 2.0 g 26 I Cholesterol Esterase 125 Units

27 ¦I Cholesterol Oxldase 125 Units

28 'I Horseradish Peroxidà~e 800 Unles

29 !1

30 I The pH is ad~usted to a pH of 5Ø
.. ~ ' ' .

39 0 a il 708-017 1 `-~

! The reagen~ system may be stored and used in the 2 ¦ form of an aqueous solution or che solution may be freeze-3 'I dried by conven~ional means and reconsti~uted wi~h wa~er 4 .j ~hen ready for use.
5 il 6 li B. AssaY - Tocal Choles~erol De~ermina~ion , -i: .
7 l Thr~-e ml of tll~ abov~ r~ag.n~ i~ mix~d with 0.025 ml 8 ¦ of serum or recons~i~u~ed serum s~andard which contains Up 9 ¦ ~o 500 mg/dl cholesterol. The reaccion is carried ouc at lO '¦ 45C for 4 ~o 5 minu~es. The absorbance oE samples a~ 525 nm ~ is measured against the reagent blank.
12'i j 13 ~¦ E~AMPI.E VI
14 ¦ Clearing in determina~ion of crea~lne phosphate kinase (CPK) j ac~ivi~y oE lipemic serum 16 il Two ml of imidazole-ace~a~e buffer, 0.1 M, pH 6.7 17 ;! containing ?auric acid diethanolamide (0.4%), pancrea~ic 18 ,I choles~erol escerase (25 U/dl), Na Cholate (0.25 g%) and l9 li thiolglycerol (20 mM), is mixed with 0.05 ml of lipemic 20 1I serum. After 15 minutes incubation at 37C, the ~urbidity 21 'I at 340 nm drops from 2.3 O.D. to 0.02 O.D. The clear sample 22 , is ~hen mixed with 1 ml of CPK reagen~ which contains 23 ¦ Crea~ine Phosphate (116-7 mM), ADP (6.7 mM), AMP (16.7 mM), ¦ ~ -24 jj EDTA (6.7 mM), NADP (6.7 mM), Hexokinase (125 U/dl), glucose 25 !¦ 6-phosphate dehydrogenase, G6PDH (100 U/dl) prepared in 2~ ~1 0.1 M imida7O1e-aceta~e buEfer, pH 6.7. The activlty of 27 ¦ CPK is monitored at 340 nm and 37C as the conventional 28 j method.
2~ '1 ,.
30 ,l `

.

~ 1~3g0~ .
708-017 !

I l¦ It should be understood by those skllled ln the 2 .¦ art that various modifications ~ay be made in tha present 3 ,' invention wichout depar~ing from the spirit and scope 4 1! chereof as described in th~ specification and defined in 5 I the appended clalms.
6 ~
7 'i 8 1 :
9 ,!
o 11 i!

,5 19 j ' I .
201~ :

~ . 254 . ~ .

26 !
271 . I , .
: 2~ I .
29 '' ,.~

,

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

l. An aqueous reagent effective in clearing turbidity of a biological sample comprising a surfactant of the formula:

wherein R is alkyl or alkenyl containing from 5 to 17 carbon atoms, x and y are each l;` and an enzyme selected from the group consisting of cholesterol esterace, lipase and mixtures thereof.
2. A reagent as claimed in claim 1, in aqueous buffered form, in which said surfactant comprises from about 0.05 g/dl to about 2.5 g/dl, and said enzyme, which is cholesterol esterase, comprises at least about 0 r 025 U/ml of the resulting formulation, said formulation having a pH in the range from about 5.5 to about 7Ø
3. A reagent as claimed in claim 2 wherein the buffer is a maleate used in a concentration of from about 0.05 M
to about 0.5 M and the pH of the resulting formulation is from about 0 r 05 to about 7Ø
4. A reagent as claimed in claim 2 which includes a solubility enhancer.
5. A reagent as claimed in claim 4 in which said solubility enhancer is selected from the group consisting of sodium cholate and sodium deoxycholate in an amount to provide about 0.1 - 0.5 g/dl of the total formulation.
6. A reagent as claimed in claim 1, in aqueous buffered form, in which said surfactant comprises from about 0.05 g/dl to about 2.5 g/dl, and said enzyme, which is a lipase, comprises at least about 1.0 g/ml of the resulting formulation, said formulatin having a pH in the range from about 5.5 to about 8Ø
7. A reagent as claimed in claim 6 wherein the buffer is a maleate used in a concentration of from about 0.05 M to about 0.5 M and the pH of the resulting formulation is from about 5.0 to about 7Ø
8. A reagent as claimed in claim 6 which includes a solubility enhancer.
9. A reagent as claimed in claim 8 in which said solubility enhancer is sodium cholate or sodium deoxycholate in an amount to provide about 0.25 g/dl of the total formulation.
10. A reagent as claimed in claim 1 wherein said surfactant is of the formula in which R is alkyl.
11. A reagent as claimed in claim 10 wherein said surfactant is lauric acid diethanolamide.
12. A reagent as claimed in claim 1 in which said enzyme is derived from animal or microbial source.
13. A reagent as claimed in claim 12 in which said enzyme is cholesterol esterase derived from animal pancreatic tissue.
14. A method for effectively clearing turbidity in a biological sample which comprises combining said biological sample with the reagent claimed in claim 1.
15. The method of claim 14 wherein said combination is in aqueous buffered form.
16. The method of claim 15 in which said biological sample is a human serum sample.
17. An aqueous reagent, effective in clearing turbidity of a biological sample, for use in the photometric assay or analysis of said sample comprising at least one surfactant of the formula:

wherein R is alkyl or alkenyl containing from 5 to 17 carbon atoms; x and y are whole number integers whose sum is no greater than 11; and an enzyme selected from the group consisting of cholesterol esterase, lipase and mixtures thereof.
18. A reagent as claimed in claim 17, in aqueous buffered form, in which said surfactant of said formula in which x + y is no greater than 5 comprises from about 0.05 g/dl to about 2.5 g/dl, and said enzyme, which is cholesterol esterase, comprises at least about 0.025 U/ml of the resulting formulation, said formulation having a pH in the range from about 5.5 to about 8Ø
19. A reagent as claimed in claim 18 wherein the buffer is a maleate used in a concentration of from about 0.05 M
to about 0.5 M and the pH of the resulting formulation is from about 5.0 to about 7Ø
20. A reagent as claimed in claim 17, in aqueous buffered form, in which said surfactant comprises from about 0.05 g/dl to about 0.5 g/dl, and said enzyme, which is a lipase, comprises at least about 1.0 U/ml of the resulting formulation, said formulation having a pH in the range from about 2.0 to about 10Ø
21. A reagent as claimed in claim 20 wherein the buffer is selected from maleate, citrate, succinate, used in a concentration of from about 0.05 M to about 0.5 M
and the pH o. the resulting formulation is from about 2.0 to about 10Ø
22. A reagent as claimed in claim 17 wherein said surfactant is of the formula in which R is alkyl and x and y are each 1.
23. A reagent as claimed in claim 22 wherein said surfactant is lauric acid diethanolamide.
24. A reagent as claimed in claim 22 wherein said surfactant is myristic acid diethanolamide.
25. A reagent as claimed in claim 22 wherein said surfactant is capric acid diethanolamide.
26. A reagent as claimed in claim 17 wherein said surfactant is of the formula in which R is alkenyl and x and y are each 1.
27. A reagent as claimed in claim 26 wherein said surfactant is oleic acid diethanolamide.
28. A reagent as claimed in claim 26 wherein said surfactant is coco acid diethanolamide.
` 29. A reagent as claimed in claim 17 in which said enzyme is derived from animal or microbial source.
30. A reagent as claimed in claim 29 in which said enzyme is cholesterol esterase derived from animal pancreatic tissue.
31. A reagent as claimed in claim 17 wherein said surfactant is of the formula in which R is alkyl and the sum of x and y is 5, and said enzyme is a lipase.
32. A reagent as claimed in claim 31 which includes polyechylene glycol p-isooctyl phenyl ether (Triton X-100 )?.
33. A method for effectively clearing turbidity in a biological sample to be photometrically assayed which comprises combining said sample with a reagent as claimed in claim 17.
34. The method of claim 33 wherein said combination is in aqueous buffered form.
35. The method of claim 34 in which said biological sample is a human serum sample.
36. The method of claim 33 in which said sample is photometrically assayed for cholesterol.
37. A reagent as claimed in claim 17, in aqueous buffered form, in which said surfactant has the formula shown comprising a mixture of lauric acid diethanolamide and epoxylated lauric acid (x + y = 5), said mixture comprising from about 0.05 g/dl to about 2.0 g/dl of the resulting formulation, said formulation having a pH in the range from about 2.0 to about 10Ø
38. A reagent as claimed in claim 2 wherein said surfactant comprises from about 0.1 g/dl to about 0.5 g/dl of the resulting formulation.
39. A reagent as claimed in claim 6 wherein said surfactant comprises from about 0.1 g/dl to about 0.5 g/dl of the resulting formulation.