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 ~.

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.

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 .