AGGLUTINATION OF INTRALIPID BY SERA OF ACUTELY ILL PATIENTS

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AGGLUTINATION OF INTRALIPID BY SERA OF ACUTELY ILL PATIENTS GEOFFREY HULMAN HENRY J. PEARSON

IAN FRASER P. R. F. BELL

Departments of Histopathology and Surgery, Clinical Sciences Building, Leicester Royal Infirmary

intensive care, 28 coronary-care staff and doctors.

patients, and 10 healthy laboratory

The results of this study led to a more comprehensive investigation of the effects of sera from 100 patients on intralipid. We chose patients with various conditions, ranging from elective surgical cases to the terminally ill.

Laboratory Investigations The serum of over half of a group of

acutely patients agglutinated ’Intralipid’ a fat emulsion based on soya bean oil designed for intravenous infusion. This reaction is probably precipitated by C-reactive protein in the presence of calcium ions. Post-mortem examinations of patients who had received intralipid showed evidence of microembolism which could have been caused by agglutination of intralipid. If this is the case then intralipid should perhaps not be given to these acutely ill patients. Summary

ill

Introduction ’INTRALIPID’ is a fat emulsion based on soya bean oil,I designed for intravenous infusion. It is widely used as a source of concentrated calories in debilitated, maltiourished patients of all ages. Some reports suggest that it may be harmful when administered to some patients. ’Lipumol’, another lipid emulsion, which is no longer available, was extensively investigated in the 1960s. It was based on cotton seed oil and was found to cream in the presence of calcium ions when added to the sera of some 2 acutely ill patients. Creaming has been investigated to only a limited extent with intralipid.3 We undertook a study of the effects of various sera on intralipid. Methods Patients An initial study was performed to determine whether creaming occurred with intralipid. We tested sera from 26 patients in

The creaming test was performed by mixing 10 ul of 2007C intralipid with 400 ul of serum. The mixtures were then incubated in capped test tubes at 370C for 24 h. The tubes were inspected for creaming 2, 6, and 24 h after mixing. Normally the mixture remains as a uniform opalescent emulsion. Creaming was judged to have occurred when a definite layer of intra lipid had formed above a clear serum layer. At 24 h each mixture was examined microscopically under high power. A normal serum/intralipid emulsion consists of countless tiny lipid particles (0’. 1-0 . 5 µm in diameter) in brownian motion. Creaming was caused by agglutination of intralipid into clumps with a diameter ofup to 200 µm. The extent of creaming was scored as +, + +, or + + + depending on the degree of agglutination. Patients whose sera cream or agglutinate intralipid are referred to as

"creamers". The albumin, total protein, and electrolyte levels of each sample measured. We compared groups by means of Student’s t test. Immunoglobulins IgG, IgA, and IgM were also determined. The relative levels of al and a2, &bgr;, and y globulins of all the serum samples were estimated by protein electrophoresis with Corning agarose strips. The level of each globulin band was determined by laser nephelometry and graded as low, normal, or raised. Groups of patients were compared by the Xtest. C-reactive protein concentrations were also determined by laser measurements of immune complexes. were

Free calcium ions are necessary to produce creaming oflipumol.2 3’ 8% citrate was added to 200 pl of ten creaming sera to determine whether calcium ions are involved in the creaming of intralipid. 200 µl of purified C-reactive protein (12 mg/dl) was also added to 200 p/l of a non-creaming serum before incubation with intralipid.

Results 11. Labro MT, Andrieu

MC, Weber M, Homberg JC A new pattern of non organ specific anti-organelle antibody detected by immunofluorescence: the mitochondrial antibody number 5. Clin Exp Immunol 1978; 31: 357-66. 12. Homberg JC, Stelly N, Andreis I, Abuaf N, Saadoun F, Andre J. A new antimitochondrial antibody (anti-M6) in iproniazid induced hepatitis. Clin Exp Immunol 1982; 47: 93-102. 13. Berg PA, Wiedmann, KH, Sayers T, Klöppel G, Lindner H. Serological classification of chronic cholestatic liver disease by the use of two different types of antimitochondrial antibodies. Lancet 1980; ii: 1329-32 14. Mackay JR, Ritts RE. WHO handbook of immunological techniques. Geneva: World Health Organisation, 1979. 15. Sayer TJ, Binder T, Berg PA Heterogeneity of antimitochondrial antibodies: characterization and separation of the antigen associated with the pseudolupus erythematosus syndrome. Clin Exp Immunol 1979; 37: 68-75. 16. Hansen M, Smith AL, Studies on the mechanism of oxidative phosphorylation. Biochem Biophys Acta 1964; 81: 214-22. 17. Smith AL. Preparation, properties, and conditions for assay of mitochondria. In: Estabrook RW, Pullman ME, eds. Methods in ensymology x. New York: Academic Press, 1976: 81-86 18. Beechey B, Hubbard SA, Linnett PE, Mitchell AD, Munn EA. A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles. Biochem J 1975; 148: 533-37. 19. Maisch B, Berg PA, Kochsiek K. Immunological parameters in patients with congestive cardiomyopathy. Basic Res Cardiol 1980; 75: 221-22 20. Baum H, Berg PA. The complex nature of mitochondrial antibodies and their relation to primary biliary cirrhosis. In: Jones EA, ed. Seminars in liver disease. vol 4. New York: Thieme Stratton Inc., 1981: 309-21 21 Miyachi K, Gupta RC, Dickson ER, Tan EM. Precipitating antibodies to mitochondrial antigens in patients with primary biliary cirrhosis. Clin Exp Immunol 1980; 39: 599-606. 22. Berg PA, Binder T. Demonstration of mitochondrial antibodies in sera from patients with chronic liver diseases reacting with two different types of complement-fixing antigens. In: Gentilini P, Popper H, Theodore U, eds Chronic hepatitis. New York: Karger, 1976: 79-85. 23. Manns MM, Meyer zum Büschenfelde KH. Mitochondrial antibody system in cholestatic liver disease detected by radioimmunoassay. Hepatology 1982; 2: 1-7.

None of the sera of the 10 healthy controls produced creaming, but sera from a high proportion of the acutely ill patients in intensive-care and coronary-care units did so (table I). In the second group of 100 patients, sera from 52 creamed TABLE I-DISTRIBUTION OF CREAMERS IN ICU AND CCU STUDIES

ICU=intensive

care

unit.

CCU=coronary care unit.

agglutinated intralipid. The conditions most commonly creaming are neoplasia; severe infections, e.g., pneumonia, septicaemia; major surgery; trauma, myocardial infarction and cerebrovascular accident; inflammatory disorders including rheumatoid arthritis; Crohn’s disease and polyarteritis nodosa; and lymphoma. The mean albumin concentration of serum producing creaming was significantly lower than that of the noncreaming group. There was no significant difference in sodium, potassium, or calcium concentrations between the two groups (table II). Immunoglobulin concentrations in the five creaming and one non-creaming sample were all normal. or

associated with

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Electrophoresis revealed that creaming sera tended to have raised al(aI-antitrypsin) and raised a2 (mainly macroglobulin and haptoglobin) bands with slightly diminished albumins. This is the protein pattern of the acute-phase reaction (table II). TABLE II-PROTEIN AND ELECTROLYTE CONCENTRATIONS OF CREAMERS AND NON-CREAMERS

the substance responsible for this phenomenon is not known. One theory was that it is a gammaglobulin, the mechanism being similar to the flocculation which occurs in the cephalincholesterol flocculation test.4 If a gammaglobulin was responsible, however, one would expect to find raised gammaglobulins in creaming sera. We found no significant difference between the gammaglobulin concentrations of creamers and non-creamers.

suggests that creaming is a property of acuteThis would explain the high proportion of creamers in intensive-care and coronary-care units. C-reactive protein is an acute-phase protein. We have found a very strong correlation between serum C-reactive protein concentration and ability to cream intralipid (see accompanying figure). Addition of C-reactive protein to a previously non-creaming serum caused it to cream intralipid. Calcium ions are implicated in the creaming process, since citrate inhibits creaming. C-reactive protein is known to agglutinate lipid suspensions in the presence of calcium ions5 and it therefore seems possible that creaming of intra lipid and other intravenous fat emulsions occurs by the same mechanism. Agglutination of intralipid is only important if it occurs to any extent in vivo. If agglutination of intralipid in the bloodstream only takes place to a minor degree it would probably be quickly cleared by the reticuloendothelial system. Agglutination to a larger extent, however, may result in lipid microembolisation. Le Veen et al. found that lipumol was cleared more quickly in creamers than non-creamers. This is attributed to microembolisation of lipumol in the tissues. He also commented that adverse reactions to lipumol (fever, shivering, precordial pains, nausea, and vomiting) only occurred in patients whose sera creamed lipumol. Intralipid has also been implicated as causing similar side-effects, though to a significantly lesser extent than lipumol. Some necropsy reports on patients who received intralipid immediately before death3,6,7 have described lipid deposition in the lungs and various other tissues including brain, kidney, and spleen. All of these patients with tissue lipid deposition were acutely ill with conditions in which one would expect to find high serum C-reactive protein levels. A possible mechanism to explain these post-mortem findings is intralipid microembolisation. Further work is in progress to assess the extent of this phenomenon in vivo and the role of in-vitro testing. Our

phase

NS=not significant. *Difference is serum albumin concentrations.

not

significance

Mean C-reactive protein concentration

if qccount is taken of ,

was

greater than

10’77

mg/dl in the creamers and less than 15 mg/dl in the non-creaming group. The higher the concentration of C-reactive protein, the higher was the percentage of creamers at that level (see accompanying figure). Of the 100 patients’ sera, all those with C-reactive protein levels of more than 10 mg/dl creamed or agglutinated intralipid. None of the ten creaming sera to which citrate was added creamed or agglutinated intralipid. The non-creaming serum to which C-reactive protein was added, before incubation with intralipid, produced creaming of intralipid. Discussion The ability of the sera of some patients to agglutinate intravenous fat emulsions has long been known.2,4 However,

study sera.

We thank Mr Mark Fuller for measuring the C-reactive protein levels, Mrs R. Aldwinckle for secretarial help, and the Department ofMedical Illustration.

Correspondence should be addressed to G. H., Department of Histopathology, Clinical Sciences Building, Leicester Royal Infirmary, P.O. Box 65, Leicester LE2 7LX. REFERENCES 1. Pelham DL. Rational 198-208.

Relation between ability of serum to agglutinate intralipid and serum C-reactive protein concentration.

Open circles represent points calculated for regression line R=0’81.

use

of intravenous fat emulsions. Am

J Hosp

Pharm

1981; 38:

2 Le Veen HH, Giordano P, Johnson A. Flocculation of intravenous fat emulsions. Am J Clin Nutr 1965; 16: 129-34. 3. Forbes GB. Splenic lipidosis after administration of intravenous fat emulsions. JClin Pathol 1978; 31: 765-71. 4. Le Veen HH, Giordano P, Spletzer J. The mechanism of removal of intravenously injected fat. Arch Surg 1961; 83: 311-21. 5 Tsujimoto M, Inoue K, Nojima S. C-reactive protein induced agglutination of lipid suspensions prepared in the absence of phosphatidylcholine. J Biochem 1980, 87: 1531-37 6. Levene MI, Wigglesworth JS, Desai R. Pulmonary fat accumulation after intralipid infusion in the pre-term infant. Lancet 1980; ii: 815-18. 7 Hessov I, Melson F, Haug A Post-mortem findings in three patients treated with intravenous fat emulsions. Arch Surg 1972; 114: 66-68.