GB2129129A - Reagent for determining beta -lipoproteins - Google Patents
Reagent for determining beta -lipoproteins Download PDFInfo
- Publication number
- GB2129129A GB2129129A GB08328420A GB8328420A GB2129129A GB 2129129 A GB2129129 A GB 2129129A GB 08328420 A GB08328420 A GB 08328420A GB 8328420 A GB8328420 A GB 8328420A GB 2129129 A GB2129129 A GB 2129129A
- Authority
- GB
- United Kingdom
- Prior art keywords
- reagent
- radicals
- lipoproteins
- same
- different
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 19
- 108010007622 LDL Lipoproteins Proteins 0.000 title abstract description 4
- 102000007330 LDL Lipoproteins Human genes 0.000 title abstract description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 8
- 150000004961 triphenylmethanes Chemical class 0.000 claims abstract description 7
- 125000002091 cationic group Chemical group 0.000 claims abstract description 6
- 125000005843 halogen group Chemical group 0.000 claims abstract description 4
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 4
- 125000004429 atom Chemical group 0.000 claims abstract description 3
- 150000001768 cations Chemical class 0.000 claims abstract description 3
- 125000001424 substituent group Chemical group 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 10
- 239000004094 surface-active agent Substances 0.000 claims description 5
- 125000000129 anionic group Chemical group 0.000 claims description 4
- 239000002738 chelating agent Substances 0.000 claims description 4
- 239000013522 chelant Substances 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 230000002452 interceptive effect Effects 0.000 claims description 2
- 125000001183 hydrocarbyl group Chemical group 0.000 claims 1
- 239000000376 reactant Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 150000001449 anionic compounds Chemical class 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 229910001412 inorganic anion Inorganic materials 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 9
- 239000000975 dye Substances 0.000 description 8
- 108090001030 Lipoproteins Proteins 0.000 description 6
- 102000004895 Lipoproteins Human genes 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 206010003210 Arteriosclerosis Diseases 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108010062497 VLDL Lipoproteins Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 208000011775 arteriosclerosis disease Diseases 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 239000004135 Bone phosphate Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000020937 fasting conditions Nutrition 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 125000001905 inorganic group Chemical group 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 150000002823 nitrates Chemical group 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical group 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
beta -lipoproteins are determined, qualitively or quantitatively, by using a colorimetric reagent constituted basically by a triphenylmethane derivative having the formula: <IMAGE> wherein the radicals R1 and R2 are the same or different and each is a halogen atom, the radicals X are the same or different and each is a hydrogen atom or an atom able to form a monovalent cation, and the radicals Y are the same or different and each is a hydrogen atom or a substituent group, together with a metal ion cheating agent, at least one inorganic anion containing compound and optionally at least one cationic or non-ionic surface another agent, of the triphenylmethane derivatives, Mordant Bleu 1 (C.I. 43830) and Mordant Bleu 29 (C.I. 43825) have proved particularly suitable.
Description
SPECIFICATION
Reagent for determining p-lipoproteins This invention relates to the use of particular triphenylmethane derivatives, referred to herein as "p-lipoprotein dyes", in the identification of -liprnprnteins. More particularly, it relates to the use of a "P-lipoprotein dye" in buffer solution and in the presence of suitable cationic or non-ionic surface active agents and of anionic groups originating from metal salts or from inorganic acids, in the qualitative or semiquantitative or quantitative determination of p-liproproteins.
Lipoproteins are known to act as liquid carriers in blood. They can be divided into four main fractions which are distinguished by the composition of the liquids carried, the carrying proteins, their size, density and electrophoretic mobility, and their antigenic characteristics. Three lipoprotein fractions can be determined in serum withdrawn under fasting conditions, namely VLDL (Very Low Density
Lipoproteins). VDL (Low Density Lipoprotein), and HDL (High Density Lipoprotein). In post-prandial serum, a fourth fraction is encountered, namely chylomicra, the presence of which in fasting subjects is pathological.
The termination of the various lipoprotein fractions is of considerable clinical interest in that they are inter alia directly related to arteriosclerosis pathology. The current methods for determining the various lipoprotein fractions are (1) electrophoresis, (2) ultracentrifugation, (3) immunological and immunoelectrophoretic methods, and (4) immunonephelometric methods. Colorimetric methods are also known, in which methods the protein concentration is obtained more or less approximately by determining the cholesterol and lipids bonded to the proteins. In particular, with regard to the LDLs, i.e.
the proteins which are currently considered to be directly related to arteriosclerosis pathology, direct spectrophotometric methods for their determination are unknown.
We have discovered that p-íipoproteins react in a specific manner with a "P-lipoprotein dye".
Thus, the present invention provides the use (in the identification of P-lipoproteins) of a "P-lipoprotein dye" chosen from triphenylmethane derivatives, in which the constant presence of the hydrogen substituents indicated in the following general formula is essential:
wherein the radicals R1 and R2 are the same or different and each is a halogen atom, the radicals X are the same or different and each is a hydrogen atom or an atom able to form a monovalent cation, and the radicals Y are the same or different and each is a hydrogen atom or a substituent group.
The present invention also provides a suitable composition comprising (i) a "B-lipoprotein dye" in a buffer solution' (ii) a chelating agent able to chelate interfering metal ions present in the sample, for the purpose of obtaining a more specific reaction; (iii) inorganic and anionic groups; (iv) and possibly cationic or non-ionic surface active agents.
Preferably, the composition comprises (a) a buffer system able to maintain the pH between 3 and 7; (b) a chelating agent able to chelate metal ions (in particular iron), particularly suitable chelating agents being citric acid and its salts, and EDTA and its salts, which can also act as buffer couples; (c) a cationic or non-ionic surface active agent, in particular cationic surface active agents such as salts or quaternary ammonium bases in which the non-hydrophobic groups are alkyl groups, cetyltrimethylammonium bromide being particularly suitable; and (d) anionic groups such as halides, nitrates, sulphates, phosphates, etc., which can be added to the mixture in the form of salts or in acid form.
The effectiveness of the method according to the present invention has been determined by chromatographically isolating the proteins of a pool of human sera which under suitable conditions give a positive reaction with a "p-lipoprotein dye", and identifying them immunologically by means of specific antisera. At the same time we verified that the other fractionated serous proteins did not give a positive reaction with the "P-lipoprotein dye" under analogous conditions.
The invention will now be illustrated by the following Examples.
Example 1
Reagent compositions for identifying lipoproteins
A reagent (A) was prepared, consisting of potassium acetate buffer (0.5 M), cetyltrimethylammonium bromide (270 mg/litre), Mordant Bleu 1 (70 mg/litre), MgCl2. 6H20 (60 g/litre), citric acid (0.026 M), and tribasic sodium citrate (0.038 M).
A reagent (B) was prepared, this reagent being the same as reagent (A) except that the Mordant
Bleu 1 was replaced by Mordant Bleu 29 in a concentration of 80 mg/litre.
Identification of p-lipoproteins in a human serum
0.1 ml of serum was added to 2 ml of the aforesaid reagent (A). The absorption spectrum of the reaction mixture thus obtained was read by a spectrophotometer against a blank constituted by 2 ml of reagent and 0.1 ml of physiological solution. A peak between 640 and 590 nm with an absorption maximum between 600 and 610 nm indicated the presence of-lipoprnteins in the serum.
0.1 ml of serum was added to 2 ml of the aforesaid reagent (B). The absorption spectrum was read in the same manner. A peak between 640 and 570 nm with an absorption maximum between 580 and 590 nm indicated the presence of p-lipoproteins in the serum.
Example 2
Reagent composition for determining lipoproteins
The following were prepared, namely a chromogenic solution analogous to that of Example 1, and a blank solution, identical to the chromogenic solution but without the "P-lipoprotein dye".
Determination of -lipoproteins in human sera
The materials in the following Table were pipetted into test-tubes.
1 Sample Sample Blank I blank Blank 2 I Serum (ml) 0.05 0.05 Blank solution (ml) - 1.5 1.5 After wating for 20 minutes, the O.D. of the sample was read at 61 5-620 nm against Blank 1 (A1), and the O.D. of the sample blank was read at 615-620 nm against Blank 2 (A2). The value A,- A2 is proportional to the /3-lipoprotein concentration in the serum.
Comparison
Eighty human sera were tested by this method. The ,B-lipoproteins were also determined in the same sera by the radical immunodiffusion method (plates supplied by Behringwerke AG). The correlation coefficient (r) of the results obtained by the two methods was 0.86.
Claims (8)
1. A colorimetric reagent for determining p-lipoproteins, comprising, as the basic reactant, a triphenylmethane derivative having the general formula:
wherein the radicals R1 and R2 are the same or different and each is a halogen atom, the radicals X are the same or different and each is a hydrogen atom or an atom able to form a monovalent cation, and the radicals Y are the same or different and each is a hydrogen atom or a substituent group.
2. A reagent as claimed in claim 1, wherein each of the radicals Y is a hydrogen atom or a hydrocarbyl group such as an alkyl group.
3. A reagent as claimed in claim 1 or 2, wherein the triphenylmethane derivative is Mordant Bleu 1 (C.l. 43830) or Mordant Bleu 29 (C.l. 43825).
4. A colorimetric reagent for determining i3-lipoproteins, comprising (a) at least one triphenylmethane derivative as defined in claim 1 , 2 or 3; (b) a chelating agent able to chelate interfering metal ions; and (c) at least one inorganic anionic group.
5. A reagent as claimed in claim 4, further comprising (d) at least one cationic or non-ionic surface active agent.
6. A method for the colorimetric determination of a /3-lipoprotein, which comprises contacting the p-lipoprotein with a reagent as claimed in any of claims 1 to 5.
7. A reagent as claimed in claim 1, substantially as described in the foregoing Example 1.
8. A method according to claim 6, substantially as described in either or the foregoing Examples.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT8223974A IT1191052B (en) | 1982-10-28 | 1982-10-28 | REAGENT FOR THE DETERMINATION OF BETA-LIPOPROTEINS. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB8328420D0 GB8328420D0 (en) | 1983-11-23 |
| GB2129129A true GB2129129A (en) | 1984-05-10 |
Family
ID=11211248
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB08328420A Withdrawn GB2129129A (en) | 1982-10-28 | 1983-10-25 | Reagent for determining beta -lipoproteins |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS5995461A (en) |
| DE (1) | DE3339042A1 (en) |
| ES (1) | ES527175A0 (en) |
| FR (1) | FR2535461A1 (en) |
| GB (1) | GB2129129A (en) |
| IT (1) | IT1191052B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2152937A (en) * | 1984-01-17 | 1985-08-14 | Bevaloid Ltd | Polymer compositions detectable in water |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU609630B1 (en) * | 1989-08-30 | 1991-05-02 | Miles Inc. | Method and composition for the assay of albumin |
| JPH0752198B2 (en) * | 1989-08-30 | 1995-06-05 | マイルス・インコーポレーテッド | Composition showing discoloration, measuring method and presence of albumin and / or concentration |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2026015A (en) * | 1978-07-24 | 1980-01-30 | Merck Patent Gmbh | Cell smear staining composition and material, and production and use thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1277592B (en) * | 1963-03-26 | 1968-09-12 | Haury Chem Fab Dr Heinz | Method for the colorimetric determination of serum ª ‰ lipoprotein |
| GB1183044A (en) * | 1966-04-05 | 1970-03-04 | Agfa Gevaert Nv | Optically Sensitized Photoconductive Recording Elements |
-
1982
- 1982-10-28 IT IT8223974A patent/IT1191052B/en active
-
1983
- 1983-10-25 GB GB08328420A patent/GB2129129A/en not_active Withdrawn
- 1983-10-27 DE DE19833339042 patent/DE3339042A1/en not_active Ceased
- 1983-10-27 FR FR8317206A patent/FR2535461A1/en not_active Withdrawn
- 1983-10-28 ES ES527175A patent/ES527175A0/en active Granted
- 1983-10-28 JP JP58201135A patent/JPS5995461A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2026015A (en) * | 1978-07-24 | 1980-01-30 | Merck Patent Gmbh | Cell smear staining composition and material, and production and use thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2152937A (en) * | 1984-01-17 | 1985-08-14 | Bevaloid Ltd | Polymer compositions detectable in water |
Also Published As
| Publication number | Publication date |
|---|---|
| IT1191052B (en) | 1988-02-24 |
| IT8223974A0 (en) | 1982-10-28 |
| FR2535461A1 (en) | 1984-05-04 |
| ES8602254A1 (en) | 1985-04-16 |
| GB8328420D0 (en) | 1983-11-23 |
| JPS5995461A (en) | 1984-06-01 |
| ES527175A0 (en) | 1985-04-16 |
| DE3339042A1 (en) | 1984-05-03 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |