Translation Series No Evaluation of the determination method of total serum lipids Colorimetric method by sulfo-phospho-vanillin reaction

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1 FISHERIES AND MARINE SERVICE 'ARCHIVES. Translation Series No Evaluation of the determination method of total serum lipids Colorimetric method by sulfo-phospho-vanillin reaction by Hideto Kushiro, F. Minakuchi, and I. Fukui Original title: Kessei soshishitsu teiryoho ni kansuru kento (Dai-2-ho). "Sulfo-Phospho-Vanillin" hanno ni motozuku hishokuho From: Rinsho Byori (The Japanese Journal of Clinical Pathology), 20(2) : , 1972 Translated by the Translation Bureau(SM/PS) Multilingual Services Division Department of the Secretary of State of Canada Department of the Brivironment Fisheries and Sarine Service Halifax, Laboratory Halifax, H.S. 1974'. 16 pages typescript

2 DEPARTMENT OF THE SECRETARY OF STATE ṮRANSLATED FROM - TRADUCI ION DE Japanese.AUTH AUTEUR TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION Pr'47À ;1) CANADA INTO - EN H. Kushiro, F. Minakuchi, and I. Fukui English SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES EeA1 3,2? TITLE IN ENGLISH - TITRE ANGLAIS Evaluation of the Determination Method of Total Serum Lipids II. Calorimetric Method by Sulfo-Phospho-Vanillin Reaction TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÉRES ROMAINS) Kessel soshishitsu teiryoho ni kansuru kento. (1Jai-2-ho). "Sulfo-Phospho-Vanillin" hanno ni motozuku hishokuho«referf-nce IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE. FOREIGN CHARACTERS. RÉFÉZENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE DU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉ:RES ROMAINS. RINSHO BYORI REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS The Japanese Journal of Clinical Pathology PUBLISHER.- ÉDITEUR Japan Society of Clinical Pathology PLACE OF PUBLICATION LIEU DE PUBLICATION Tokyo, Japan YEAR ANNÉE DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES nt, REQUESTING DEPARTMENT Environmimt TRANSLATION BUREAU NO MINISTRE-CLIENT NOTRE DOSSIER Nt) EIRANCH OR DIVISION TRANSLATOR (INmALs) Fisheries Service SM / DIRECTION OU DIVISION TRADUCTEUR (INITIALES) PERSON REQUESTING DEMANDÉ PAR Allen T. Reid OCT YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST DATE DE LA DEMANDE UNEDITED TRI-d\ISLAïlON For information only s TRADUCTION NON furtiv:e. Informa!ion souiom SOS-ZOO-10-8 (REV. 2/88)

3 DÉPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS MULTILINGUAL SERVICES DIVISION CANADA DIVISION DES SERVICES MULTILINGUES client's No. DEPARTMENT DI VISION/BRANCH CITY N 0 DU CLIENT MINISTRE WVISION/DMECTION VILLE Environment Fisheries Service Halifax N - S. BUREAU NO, LANGUAGE TRANSLATOR (MI TIALS) N DU BUREAU LANGUE TRADUCTEUR (INITIALES) Japanese SM/PS 11 Oct., 1974 Evaluation of the Deteirminatidn Method of Total Serum Lipids II. Colorimetric Method by Sulfo-Phospho-Vanillin Reaction OCT Hideto Kushiro*, Fusako Minakuchi*, and Iwao Fukui, M.D.* There are several methods used to quantitatively P. 1. determine the total lipids in Serum. They are widely classified as the gravimetric, oxidimetric, nephelometric, volumetric, pigment bonding, and calculation methods and calorimetric UJ dethod using a sulfo-phospho-vanillin reaction Wiereafter L r "(5 g abbreviated as &V method) 1. tr,.2 0 Among. them the SPV method is especially noticeable n,2 n W.! Us f.,) 411 cf: 9. because its superb conditions regarding smallness of volume of serum A required, speed and precision. The procedure of quantitative analysi by the SrV method is composed of two steps: Sulfonation of serdm lipids by sulfuric acid and then color development by the * Central laboratory for Clinical Investigation, Kyoto Prefectural University of Medicine, Kyoto.

4 2 SPV reaction 2,3. There are several modifications of this method reported by Frings, Wada 5, Okuda 6, and Matsumiya. In our previous paper 8, we compared the values of total serum lipids determined by the gravimetric, oxidimetric, and SPIT methods: we found that these methods gave different results. In the present paper we investigated the difference in the coloration of the lipid components in serum by the SPV reaction. We compared the experimental values obtained by the SPV method and calculation method and discussed the accuracy of the experimental values obtained by the SPV method. I. Investigation methods and materials The total lipids in serum specimens and the lipids in the standard solutions were determined using Frings method 4. Serum cholesterol was meabured using the extraction method reported by Zak which is based on the Killiani reaction 9. Phospholipids were studied using a modified Hoeflmayer- Fried method 10, triglycerides wl*ing a modified VanHandel- Zilversmit-Fukui method ll, and.fre e. fatty acids (FFA, for short) using an improved Itaya-Ui method 12.

5 3 Standard lipids were as follows: olive oil (olive oil for the determination of lipase, purchased from N.B.C. ); cholesterol (from Daiichi Chemicals); phoapholipids ( DL- D^-- lecithin, from Sigma); triglycerides (tripalmitin, from Iiodak) ; and FFA (palmitic acid, from Hanni Cnef-nica:Ls). ConmmercialZy available control serum used in this experiment was the Special Clinical Chemistry Control Serum (Lot No. 3699E010A1) purchased from Hyland. The absorption spectrum was measured using a riitachi Ltd. Model.101 spe c trophotometer. - Other properties were measured using/,shimazu Seisakusho Ltd. Boschro,n-type diffraction photoelectric-colorimeter. xt. Results A. AbsoKptior_ Sp2ctÿu1n The absorption spectra of various lipid specimens colored by the SPV reaction are shown in Fi g. 1(a), The curves for serum, commercial control serum, olive oi7_, phospholipi ds, triglyce-rides and FFA have a peak (Xmar ) at P mlt and c'rlolester o1. at 540 rju.

6 4 B. Linearity and sonsitivitz. The calibration curves of these specimens were linear up to a concentration of 500 mg/di and all of them started from the origin. Fig. 1(b) shows these results. O.D Serum Chol est erol Olive oil (h) Cholesterol Olive oil Control serum FFA PL TG (mil s; 5S0 600 Fig, 1(a) Absorption spectra of specimens. FFA: free fatty acid; PL: phospholipids; TG: triglycarides tag/d/ Concentration of Lipids Fig. 1(b) Linearity and sensitivity. However, their sensitivities differ from each other greatly: the absorption coefficients of these lipide at a concentration of 500 mg/dl were for cholesterol, for olive oil, for FFA, for phospholipids, and for rates triglycerides. Thevof colorationfor FFA, phospholipids and triglycerides were very low, they were only 1/3 of those for

7 cholesterol and olive oil. C. Sulfonation Sulfonation of lipids O.D [ Semm Olive oil Was conducted at 100 C using sulfuric acid and the time o nr Cludeeerot centre l :17G FFA PL Time of Sulfonikation 30 min. 10, 15,.20, and 30 minutes Fig. 2 Time of sulfonation and rate of coloration. -after the reaction was initiated. The results are shown in Fig. 2. The rates of coloration for serum, control serum and olive oil reached their maximum in 20 minutes and decreased at 30 minutes, while the rate for cholesterol reached its maximum in 15 minutes and was stable and constant after this. Those for triglycerides, FFA, and phospholipids, increased linearly with time. D. SPV reaction The most suitable concentration of lhospho- vanillin reagent and the dolor change according to time of trie specimen were studied.

8 6 1. Phospho.Wanillin reagent. a) Concentration of vanillin: Vanillin solution of five different concentrations, 0.2, 0.4, 0.6, 0.8 and 1.0 w/v% e were prepared. Each of them was mixed with an 85 w/v%* phosphoric acid. The rate of coloration for serum, commercial control serum and the lipid standards using these phospho-vanillin reagents were then studied. For all of these specimens the rates reached their maximum at about 0.8 w/v%: however, their rate of increase depended on each particular specimen Change of O.D. Color with time The time change r Serum Oh,/ e on 0.1a Of color, with time which was caused by the UPV reaction, depend- ed on each particular specimen. This fact can be seen in Fig. 3. E. Accuracy_2f_nnsurements Fig. 3. TG FFA. PL 1 [ M mm The Time change of color. The accuracy of measurements (C.V.) by the simul- taneous measurements was. èstimated as ± 3.7% for serum, _ * Translator's note: v/v% in original must be a typographical error for w/v'/b.

9 7 -I-3.e for olive oil, 44.2% for cholesterol, +2.6% for triglycerides, ± 30% for phospholipids, and for FFA. F. Correlation between the SPV method and calculation method. The contents of cholesterol, phospholipids, triglycerides and FFA in serum were determined separately using the aforesaid methods 9, 10, 11, 12 and then the total lipid concentration was calculated from the values of these lipid fractions. Twenty-one serum specimens, obtained from patients when they had empty stomachs, were investigated using the above-mentioned method. The'rcsults of the calculation method were compared with those of the SPV method (cf. Table 1). The correlation between the calculation method and SPV method can be represented by the coefficient of correlation (n. The coefficient was 0.76 and 0.83 for olive P. 137 oil and used as control serum, respectively, when they were standard in the SPV method. The mean values of the experimental results are as follows: SPV method (olive oil as standard): 567 mg/di; SPV method (control serum as standard; reading by the gravimetric method: 666 mg/di; SPV method (control

10 8 Table 1. Comparison of the results obtained by the SPV method and the calculation method. Standard material SPV method Calculation Coefficient of for SPV method (mg/di) method (mg/di) correlation (fl 1. Olive oil Control serum:. reading by gravi ,83 metric method reading by calcu lation method Note: n = 21; double measurements serum as standard; reading by the calculation method): 498 mg/d1; Calculation m2thod: 500 mg/d1. The concentration of lipids measured and used in the calculation method were 198 mg/di for cholesterol, 164 mg/di for phospholipids, 128 mg/d1 for triglycerides, and 10 mg/di for FFA. III. Discussion The color formation in the SPV method may be caused by the reaction between aromatic aldehyde and decomposition

11 9 products of lipid components, having unsaturated bonds (>0 = 0<), These decomposition products may be lipid fragments, which were combined to keto-compounds through a peroxide formation process. Details of the reaction mechanism involved in this method are not clear at present. Frings and Dunn stated in their paper 4 that except for lipids, only small amounts of substances which react with the phospho-vanillin reagent are contained in serum; the :SPIT method, therefore, can be directly used for the determination of lipids in serum. Apart from the effectof thesubstances other than lipids in serum, we must consider the difference of effects between various lipid components themselves. Serum is composed of lipids such as cholesterol, cholesterol esters, phospholipids, triglycerides, FFA. Their chemical structures differ from each other greatly. Therefore, when these lipids are determined by the SPV method, we must consider the reaction of each lipid carefully or it will affect the accuracy of measurements. The rates of coloration for various standard solutions of lipids, all having a concentration of 500 mg/di,

12 10 were investigated using the saine measuring conditions reported by Frings and Dunn 4. The rates of coloration for cholesterol and olive oil were large and those for phospholipids and triglycerides were small; the former are about three times larger than the latter. We tried to find suitable conditions with which all lipids show the same rate of coloration. To this end, various conditions such as sulfonation time, concentration of phospho-vanillin reagent, SPY reaction time, were studied: however, we could find no such condition. We must, therefore, recognize that there is a certain limit in the accuracy of the determination of the total amount of lipids in serum by the SPV method. The selection of standard material is very important in this method. In the previous paper 8, we compared the values of total serum lipids obtained by gravimetric method, oxidimetric method and SPY method. From a survey of various references, we concluded that the value obtained by the gravimetric method is the true value; we reported that the commercial control serum calibrated by the gravimetric

13 11 method is more suitable than olive oil as standard material for the SFV method. However, it is still questionable if the value obtained by the gravimetric method is the "true value" in the strict sense of the word: for the time being, "standard value" might be a more suitable word for i t. In the calculation rnethod, each lipid component in the serum such as cholesterol, phospholipids, triglycerides and FFA is determined separately and the total amount of 1.ipids is obtained from the values of lipids fractions. We thought 't-his method gave a more accurate value than did the gravimetric method. Therefore, we compa r-ed the values obtained by the SPV method with those of the calculation tnethad: these results were used to select the best standard material for the SPV ne thod. It beca:ne clear that with the SPV:method, the commercial control serum the concentration of which is calibrated by the calculation method, is more suitable than olive oil, the present standard: the commercial control serum gives a closer value to the calculated one than does olive oil and also gives a better correlation coefficient than the latter.

14 12 IV. SIti,m2.Em The lipid components in serum react differently in the SPV reaction and no reaction condition which gives the same rates of coloration for these components was found. Therefore, we must recognize that there is a certain limit in the accuracy of the value of the total serum lipids ob- tained by the SPV method. P. 138 After thorough consideration of this fact, if commercial control serum (its concentration must be cali-, brated by the calculation method), which is a heterogeneous system having nearly the same components as the specimen to be determined, is used as the standard in the SPV method, then an experimental value which is very close to that found by the calculation method may be obtained. Moreover, if a commercial control serum is used as a standard, it is possible to make the most of the advantages of the SPV method, such as the small amount of specimen required, speed and accuracy: wa believe the proposed method is suitable for the daily clinical tests on the total serum lipids which must be conducted in large numbers.

15 13 References 1) Fukui, I. and Kushiro, H.: "Total lipids",,jap. J. Clinical Pathology. Special issue No. 19 ("The Practical side of quantitative analysis of lipids"): Pp 38_.719 (1972). 2) Zollner, M. & Kirsch, Ti.: über die quantitative Bestimmunô von Lipoidin (1`rTi^-xomethode) mittels der vielen natürlichenl Lipoidin (allen bekannten Piasmalipoiden)' gemeinsamen Sulfophosphovanillin Reaktion, Z. Gesam. Exp. Med., 135 : 546^-561, ) Drevon, B. &: Schmit, J.14 f.: La reactionl sulfo-phospho-vanillique dans letude des lipides seriques, Bull. Trav. Soc. Pharm., 8: 173-r178, ;^rc 7) 1D ; f M. 4) :-rings, C. S. & 1)unn; R. T. : A colorimetric method for determination of total serum for translation, see p. 16 lipids based on the sulio - phosoho vanillin reaction, Am. J. Clin. Path., 5389^91, ) i^lada, M., et al. :"Determinati on of total serum lipids and its'clinical significance. (1) Determination by SPV method.", Jap. J. Clinical Pathology, 18, pp ^, (19.10), 6) Okuda, K., et al.: "Colorimetric total serum lipids determination method (Part 2). Simplification of the operation in the reaction and minimization of specimen. Jap, J. Clinical Pathology., 18 (Supplement), 42, (1980 Translator's note: 1980 must be a typographical error.

16 14 7) Matsumiya, K., et al: "Rapid colorimetric method (sulfo-phospho-vanillin) for the determination of total serum lipids." Jap. J. Clinical pathology, 19, pp , (1971). 8) Kushiro, H., et al.: "Evaluation of the determination methods for total serum lipids. 1. Comparison of total serum lipids values obtained by gravimetrie method and oxidimetric method and SPV method." Jap. J. Clinical Pathology, 19, pp , (1971). 9) Fukui, 1. and Kushiro, H.: "Practical Biochemistry: Cholesterol", edited by Kosakai, N. and Tomita, H., published by Igakushoin (Tojcyo), pp , (1970), 10) Kushiro, H. and Fukui, "Evaluation of the measuring method of phospholipids in serum (Hoeflmayr-Fried method)." Jap. J. Clinical Pathology, 15, pp (1967). 11) Fukui, I: "Measurement of MDS KoWa and neutral fat." Kowa Medical Magazine, No. 6: pp 14-20, (1964). 12) Kushiro, H. et al.: "Evaluation of colorimetric serum free fatty acid-s determination methods, VII.- On the modified Itaya-Ui method." Jap. J. Clinical Pathology 18, pp , (1970).

17 15 Re.fererlces (-transj.iteration) 1) I. Fukui, H. Kushiro: "Soshishitsu". Rinsho Byori, Rinji--^okan, Tokushu 19-go (Shishitsu teiryo no jissai), 38-51, (1972). M. Wada, hoka: " K.essei soshishitsu no sokutei to rinshoteki igi (1), SPVpha.nno ni yoru shin-sokuteiho no kento." Rinsho Byori, 18, , (1970). 6) K. Okuda, hoka: "Kessei soshishitsu no hishoku teiryoho (dai--2--ho), hannososa no kariika to kentai no biryoka." Rinsho Byori, 18 (hosa-l-su) 42,. (1980* ). 7) K. l';la-tsumiya, hoka: " Suifo--.Phosbl?o-Va.ni.llin ni yoru kessei soshishitsu biryo-teiryoho." Rinsho.Byori, 19, , (1971). Ii. Kushiro, holça: "Kessei soshishitsu teiryoho ni kansuru kento ( dai--1 -po, juryoho., sankaho narabini SPV-ho ni yoru toiryochi no hikaku)". Rinsho Byori, 19, , (1971). 9) I. Fukui-, H. Kushiro-. "koresuteroru, Seikagaku Jisshu, H. Konishi, H. Tomita henshu, Igaku Shoin (Tokyo), (1970), P. 135-'140 10) H. Kushiro, I. Fukui: "Kessei rin-shishitsu soku-tei.ho * i'ran:,la-lor' s no-te: 1980 mustl be a-t,ypoffraphical error.

18 o 16 ni kansuru kento (Hoeflmayr-Fried ho ni tsuite)." Rinsho Byori, 15, 57-62, (1967). 11) 1. Fukui: "MDS Kowa to chusei shibo no sokutei ni tsuite." Kowa Iho, dai-6, 14-20, (1964). 12) H. Kushiro, hoka: "Kessei yuri-shibosan hishoku teiryoho ni kansuru kento (dai-7-ho, itaya, Ui kairyoho ni tsuite)." Rinsho Byori, 18, , (1970). 2. Milner, M. & Kirsch, K. On the quantitative determination of lipoidin (micro-method) by means of the sulfophospho-vanillin reaction, the latter being common to many natural lipoids (all known plasma lipoids).

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish FISHERIES AND MARINE SERVICE ARCHIVES Translation Series No. 3637 Investigation of the lipid composition of several species of fish by A.G. Rudenko, and V.I. Lisovskaya Original title: Issledovanie sostava