3
Clin. Biochem. 9 (2) 96-98 (1976) Bilirubin Quantitation with Lipemic Plasma GEORGE CHAN, KATHY MERRILLS and DAVID SCHIFF Department of Pediatrics and Obstetrics and Gynecology, University of Alberta, Edmonton, Alberta (Accepted December 1976) CLBIA 9, (2) 96-98 (1976) Clin. Biovhem. Chan, George, Merrills, Kathy, and Schiff, David DepartmeNt of Pediatrics and Obstetrics and Gyna- eology, University of Alberta, Edmonto~z, Alberta BILIRUBIN QUANTITATION WITH LIPEMIC PLASMA 1. In the presence of lipemia, the estimation of BR by diazo method is variable and hence unre- liable. 2. The' estimation of BR in lipemic plasma by the use of the A-O bilirubinometer yielded BR levels which were consistently lower than theoretical values. 3. By regression analysis of the percent error in BR estimation, (using the A-O bilirubinometer) and TG Concentrations, a straight line is obtained. Bascd upon this line, a correction factor for plasma BR concentration in the range of 5-25 mg/dl can be obtained if the degree of lipemia is known. THE USE OF INTRAVENOUS FAT as a source of nutrition for low birth weight infants is becoming more popu- lar "). It was observed during a pilot study to assess the newborn's ability to utilize Intralipid"-' '~ (a soy- bean fat emulsion), that when the infant's plasma became either grossly or biochemically lipemic, esti- mation of BR levels by the diazo reaction was vari- able. The present study was undertaken to establish the validity of plasma BR concentrations under these circumstances using a modified diazo reaction"' and an AO bilirubinometer '5~. MATERIALS AND Mm'HODS To assess the correlation between BR concentrations using direct spectrophotometry (AO bilirubinometer, Model 10200) and the diazo reaction, BR concentrations in non-lipemic plasma obtained from 41 newborn infants was determined by both methods. The BR concentration ranged from 1.5 to 24.0 mg/dl. The bilirubinometer is supplied with a stable artificial standard which simulates the absorbance of BR at 461 rim. The instrument is calibrated with this standard just prior to the determi- nation of BR. From the diazo reaction, BR standards were prepared with the commercially available pigment (sigma) without further purification. 10 mg. of BR was weighed out accurately with a Metler microbalance and dissolved in 0.5 ml of 0.1 N NaOH. When the BR was completely in solution, 0.5 of buffer {0.45% Na~CO.~ and 0.45% NaC1, pH 7.8) was added and mixed well. This preparation was kept in the dark and was stable for several hours but was usually used within the hour. A Abbreviations: BR -- Bilirubin; TG -- Triglycerides. Correspondence: Dr. D. Schiff, Dept. of Pediatrics, University of Alberta, Edmonton, Alberta T6G 2G3, standard curve was constructed by diluting the BR solu- tion with 3 g/dl human albumin made up in isotonic phosphate buffer, pH 7.4. Linearity of concentration versus absorbance was maintained from 0 to 24 mg/dl when tests were performed in a Beckman DB spectropho- tometer. To assess the effect of lipemia on BR estimations, BR standards (5 to 25 mg/dl) were prepared as described above and were artificially made lipemic with Intra- lipid 10%*. In duplicate, 20 ~l of serial dilutions of Intra- lipid with concentrations of TG from 0.5 to 10 mg/dl were mixed into 200 ~1 of the different BR standards to give final TG concentrations of 50, 100, 250, 500, 750 and 1000 mg/dl. Volume corrections were made in control standards by addition of 20 .~l saline. BR concentrations were again determined using the two methods. RESULTS Fig. 1 compares the results of BR concentrations in the 41 hyperbilirubinemic non-lipemic plasma ob- tained by the bilirubinometer and the diazo reaction. There is an excellent correlation between the two methods with a regression coefficient of 0.99 and the slope of the regression line is given as y ---- -0.054 -}- 1.006 x. The effects of iipemia on BR levels by the two meth- ods are shown in Tables 1 and 2. Further differentia- tion of the data (Table 3, 4) showed that at moderate lipemia (TG up to 50 mg/dl), results obtained by both methods correlate well with theoretical and control *Pbarmacia (Canada) Ltd. 30 ~'25 Z 20 i o z N 10 ,y ,_,,1 r~ 5 0 l, X K X X DIAZO REACTION - BILIRUBIN mg/dl. ~5 ~o Fig. 1 -- Correlation of plasma bilirubin concentration iN noel lipemie specimens between the bilirubinometer and thc dia=o reaction, n ~ 51, r ~ 0.99, y = 0.055 Jr- 1.006 ~.

Bilirubin quantitation with lipemic plasma

Embed Size (px)

Citation preview

Page 1: Bilirubin quantitation with lipemic plasma

Clin. Biochem. 9 (2) 96-98 (1976)

Bilirubin Quantitation with Lipemic Plasma G E O R G E C H A N , K A T H Y M E R R I L L S and D A V I D S C H I F F

D e p a r t m e n t o f P e d i a t r i c s a n d O b s t e t r i c s a n d G y n e c o l o g y , U n i v e r s i t y o f A l b e r t a , E d m o n t o n , A l b e r t a

( A c c e p t e d D e c e m b e r 1976)

CLBIA 9, (2) 96-98 (1976) Clin. Biovhem.

Chan, George, Merrills, Kathy, and Schiff, David

DepartmeNt of Pediatrics and Obstetrics and Gyna- eology, Universi ty of Alberta, Edmonto~z, Alberta

BILIRUBIN QUANTITATION WITH LIPEMIC PLASMA

1. In the presence of lipemia, the estimation of BR by diazo method is variable and hence unre- liable.

2. The ' estimation of BR in lipemic plasma by the use of the A-O bilirubinometer yielded BR levels which were consistently lower than theoretical values.

3. By regression analysis of the percent error in BR estimation, (using the A-O bilirubinometer) and TG Concentrations, a straight line is obtained. Bascd upon this line, a correction factor for plasma BR concentration in the range of 5-25 mg/dl can be obtained if the degree of lipemia is known.

THE USE OF INTRAVENOUS FAT as a source of nu t r i t i on for low b i r th weigh t in fan ts is becoming more popu- lar "). I t was observed dur ing a pilot s tudy to assess the newborn 's abi l i ty to uti l ize Intralipid"- ' '~ (a soy- bean f a t emuls ion) , tha t when the in fan t ' s p lasma became e i the r grossly or biochemically lipemic, esti- mat ion of BR levels by the diazo react ion was var i - able. The present s tudy was under taken to establ ish the val id i ty of plasma BR concent ra t ions under these c i rcumstances us ing a modif ied diazo r eac t i on" ' and an AO b i l i rub inomete r '5~.

MATERIALS AND Mm'HODS

To assess the correlation between BR concentrations using direct spectrophotometry (AO bilirubinometer, Model 10200) and the diazo reaction, BR concentrations in non-lipemic plasma obtained from 41 newborn infants was determined by both methods. The BR concentration ranged from 1.5 to 24.0 mg/dl. The bilirubinometer is supplied with a stable artificial standard which simulates the absorbance of BR at 461 rim. The instrument is calibrated with this standard just prior to the determi- nation of BR. From the diazo reaction, BR standards were prepared with the commercially available pigment (sigma) without fur ther purification. 10 mg. of BR was weighed out accurately with a Metler microbalance and dissolved in 0.5 ml of 0.1 N NaOH. When the BR was completely in solution, 0.5 of buffer {0.45% Na~CO.~ and 0.45% NaC1, pH 7.8) was added and mixed well. This preparation was kept in the dark and was stable for several hours but was usually used within the hour. A

A b b r e v i a t i o n s : B R - - Bilirubin; TG - - Triglycerides.

Correspondence: Dr. D. Schiff, Dept. of Pediatrics, University of Alberta, Edmonton, Alberta T6G 2G3,

standard curve was constructed by diluting the BR solu- tion with 3 g/dl human albumin made up in isotonic phosphate buffer, pH 7.4. Linearity of concentration versus absorbance was maintained from 0 to 24 mg/dl when tests were performed in a Beckman DB spectropho- tometer.

To assess the effect of lipemia on BR estimations, BR standards (5 to 25 mg/dl) were prepared as described above and were artificially made lipemic with Intra- lipid 10%*. In duplicate, 20 ~l of serial dilutions of Intra- lipid with concentrations of TG from 0.5 to 10 mg/dl were mixed into 200 ~1 of the different BR standards to give final TG concentrations of 50, 100, 250, 500, 750 and 1000 mg/dl. Volume corrections were made in control standards by addition of 20 .~l saline. BR concentrations were again determined using the two methods.

RESULTS

Fig. 1 compares the resul ts of BR concent ra t ions in the 41 hyperb i l i rub inemic non-l ipemic plasma ob- tained by the b i l i rub inomete r and the diazo react ion. There is an excellent corre la t ion between the two methods with a regress ion coef f ic ien t of 0.99 and the slope of the regress ion line is given as y ---- -0.054 -}- 1.006 x.

The ef fec ts of i ipemia on BR levels by the two meth- ods are shown in Tables 1 and 2. F u r t h e r d i f f e r en t i a - tion of the data (Table 3, 4) showed tha t at modera te l ipemia (TG up to 50 m g / d l ) , resul ts obtained by both methods corre la te well wi th theore t ica l and control

*Pbarmacia (Canada) Ltd.

30

~'25 Z

20

i

o z N 10 , y

,_,,1

r ~

5

0 l,

X K

X

X

DIAZO REACTION - BILIRUBIN mg/dl. ~5 ~o

Fig. 1 - - Correlation of plasma bilirubin concentration iN noel lipemie specimens between the bilirubinometer and thc dia=o reaction, n ~ 51, r ~ 0.99, y = 0.055 Jr- 1.006 ~.

Page 2: Bilirubin quantitation with lipemic plasma

BR QUANTITATION WITH LIPEMIC PLASMA

TABLE I. EFFECT OF LIPEMIA ON BILIRUBIN DETERMINATION BY THE DIAZO REACTION

TOTAL BILIRUBIN - - mg/dl

97

TG TG TG TG TG TG Th~retical Conffol 50mg% 1~ mg% 250mg% 500mg% 750mg% lO00mg%

5.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.0 5.3 3.5 5.0 5.0 5.5 2.0 4.8 5.3 4.5 4.5 5.5 8.0 6.0

10.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 10.0 8.0 9.0 8.0 10.0 10.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 10.0 8.5 9.5 9.5 4.5

7.0 9.5

15.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 14.0 13.5 12.0 12.0 15.0 13.0 15.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 14.0 12.5 13.5 13.5 15.0 11.5

20.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 19.0 20.0 17.0 19.0 19.0 22.5 20.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5 20.0 18.0 16.0 18.5 16.5 21.0

25.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.5 24.0 25.0 24.0 2 1 . 5 2 2 . 5 28.5 25.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.5 24.0 24.0 25.0 20.5 22.0 27.5

TG = Triglyceride

~alues to wi th in 2% with s t andard deviat ions between duplicates of 0.25. However, as l ipemia in the samples increases, discrepancies f rom theoretical values were noted in both methods.

D I S C U S S I O N

The above data demons t ra ted once aga in the good correla t ion between BR results by the diazo react ion

o e ~ e ~

b--

Z

¢_)

h J

- B

-10

-5

0 o i ' 10

TRIGLYCI~IDE CONCENTRATION mgldl x 100 Fig. 2 - - B i l i rub in correct ion curve for l ipemia .in AO bi l irubinometer , model 10200. E f f e c t i v e range of bil irubln, 5 to ~5 m g / d l . Regress ion of line y = 5 T 0.8 x.

and the b i l i rub inomete r in non-l ipemic and mildly lipemic plasma. Sl ight t u rb id i ty does not seem to in ter - fere with the format ion of azobi l i rubin color and ab- sorbances at 545 nm are correctable by the blank in the diazo method. The b i l i rub inomete r utilizes undi luted plasma and measures the color of the BR concentra- t ion at 461 nm when compared to an ar t i f ic ia l s tand- ard. Tu r b i d i t y caused by l ipemia a f f e c t s ' t h e level of l ight bu t not the relat ive rat io of l ight in tens i t ies to the two photo-detectors.

At TG levels of 100 mg/d l or higher, the diazo reac- t ion yielded resul ts tha t averaged 7.5 to 13% f rom expected values. The high s tandard er ror of the mean and the high s tandard deviat ion between duplicates makes the test unrel iable as the sample becomes more lipemic, and therefore this method is not recom- mended for use under such circumstances.

On the other hand, with the b i l i rub inomete r TG levels of 100 to 1000 mg/d l also caused the BR con- cent ra t ions to be 5 to 13% less than theoret ical values. However, the level of l ight in tens i ty affected, seems to be directly related to the l ipemia in the sample. The low s tandard er ror mean of the percentage e r ror and s tandard deviat ion between duplicate samples indicate consistency of BR de te rmina t ion under grossly lipemic condit ions u t i l iz ing this methodology. A regression analysis of the percent er ror and TG concent ra t ions from 100 to 1000 mg/d l yielded a s t r a igh t line (Fig. 2). Based upon this line, a correc- t ion factor for plasma BR concentra t ion in the range of 5 to 25 mg/dl can be obtained if the degree of l ipemia is known. TG levels in lipemic plasma can easily be de termined by nephelometry r equ i r i ng only 50 /zl of plasma ~6).

I t is now being recommended tha t fat be gireen as a cont inuous infus ion to the newborn i n f a n t ~'). The in- fusion of fa t is not wi thout a t t endan t complications'3~.. Therefore, the es t imat ion of BR and when possible free fa t ty acids and reserve a lbumin b ind ing capac- ity ~) mus t cont inue on in fan t s receiving fa t pa- rentera l nu t r i t i on . An accurate assessment of plasma BR can be obtained with the b i l i rub inomete r provid- ing appropr ia te corrections for l ipemia as suggested above are made.

Page 3: Bilirubin quantitation with lipemic plasma

98 CHAN, M E R R I L L S , S C H I F F

TABLE 2. EFFECT OF LIPEMIA ON BILIRUBIN DETERMINATION BY THE BILIRUBINOMETER

T O T A L B I L I R U B U N - - mg/dl

TG TG TG TG TG TG Theoretical Control 5 0 m g % 1 ~ mg% 250mg% 500 mg% 7 ~ mg% 1000mg%

5.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 5.3 5.0 5.0 4.5 4.5 4.5 5.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 5.5 5.0 5.0 4.3 4.5 4.5

1 0 . 0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

15.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 0 . 0 . . . . . . . . . . . . . . . . . . . . . . . . .

20.0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 5 . 0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

25.0 . . . . . . . . . . . . . . . . . . . . . . . . . . .

9.5 10.0 9.0 8.4 9.0 9.0 8.5 9.5 10.0 9.5 9.0 9.0 9.0 9.0

15.0 14.3 13.0 13.0 13.0 13.8 13.2 15.3 14.0 13.8 13.5 13.0 13.0 13.0

20.0 19.0 20.0 19.0 18.5 16.7 16.0 19.5 19.5 19.0 18.5 18.5 17.5 17.0

24.0 24.0 2~.0 23.5 22.5 22.5 21.5 24.5 24.0 2~,.0 24.0 23.0 22.5 21.5

TG = Triglyceride

TABLE 3, EFFECT OF LIPEMIA ON THE CHANGE IN BILIRUBIN CON- CENTRATION EXPRESSED AS PERCENT FROM THEORETICAL VALUES

Diazo Reaction Bilirubinometer

Triglyceride % error from % error from mg/dl theoretical -4- SEM theoretical 4- SEM

0 - 1.9 4- 0.6 - 1.7 ± 1.1 50 - 1.5 4- 1.5 - 1.2 4. 1.7

100 -13.1 4- 3.5 - 5.3 4. 1.4 250 - 9.4 4. 2.3 - 7.1 4. 1.5 500 - 9.0 4. 3.0 --10.5 4. 0.8 750 --12.9 4. 6.3 -11 ,0 4- 0.8

1000 - 7.5 4. 8.7 -13.4 4. 1.0

TABLE 4. EFFECT OF L1PEMIA ON STANDARD DEVIATION BETWEEN DUPLICATES

Diazo Reaction Bilirubinometer

Tr!glycerides S.D. between S . D . between mg/dl duplicates mg% duplicates (mg%)

0 0.28 0.26 50 0.10 0.20

100 0.85 0.54 250 0.69 0.27 500 0.73 0.17 750 2.20 0.36

1000 1.65 0.37

ACKNOWLEDGEMENT

T h i s w o r k w a s s u p p o r t e d by a g r a n t , n u m b e r 609 1023 209, f r o m t h e D e p a r t m e n t of N a t i o n a l H e a l t h & W e l f a r e , O t t a w a , Can ad a .

R E 3 ~ E R E N C E S

I. Cashore, W. J., Sedagha t ian , M. R. and Usher , R. H. (1975). Pediatrics, 56: 8-16.

2. Andrew, G., Chan, G. and Schiff , D. (1976) J. Pediatrics 88 : 273-278.

3. Andrew, G., Chan, G. and Schiff , D. (1976) J. Pediatrics 88 : 279-284.

4. Mart inek, R. G. (1966). Clin. Chbn. Acta. 13: 161-170. 5. Amer ican Optical Corporat ion, Scient i f ic I n s t r u m e n t

Division, Buffalo, N.Y. 6. Carlson, L. A. and Rossner , S.(1972). Scand. J. C/iu.

Lab. Invest. 29: 271-280. 7. Chan, G. and Schiff , D. F u r t h e r clinical evaluat ion

of the Sephadex G-25 elution technique in the manage- ment of the hyperbi l i rubinemic infant . In te rna t iona l Bilirubin Symposium, May 1974, Je rusa lem, Israel . Bir th Defect Series. In Press .