Defective lipid disposal mechanisms during bacterial infection in rhesus monkeys

Defective Lipid Disposal Mechanisms During Bacterial Infection in Rhesus Monkeys Robert

L. Kaufmann,

Charles

F. Matson,

Mechanisms producing hypertriglyceridemia during bacterial sepsis have not been well defined. In this study lipid disposal mechanisms were assessed in 76 infected and 19 control male rhesus monkeys by the ability to dispose of triglycerides after: (1) oral lipid loading; (2) intravenous lipid loading; and (3) by lipolytic enzyme activity tests as measured by postheparin lipolytic activity (PHLA). Studies were performed both before and 48 hr after intravenous inoculation with either Salmonella typhimurium or Diplococcuspneumoniae when illness was uniformly severe and fasting serum triglyceride elevations were increased maximally. S. typhimurium-infected monkeys demonstrated significant fasting hypertriglyceridemia (p < O.OOl), reduced clearance

Alan H.

Rowberg, and

William

R. Beisel

of orally and intravenously administered lipid and markedly reduced PHLA. During this gram-negative sepsis, mild lethargy, slight diarrhea, and a 2% mortality were observed. During D. pneumoniae sepsis, average fasting triglyceride concentrations were slightly, but not significantly elevated. While oral lipid clearance was impaired, intravenous lipid clearance was unimpaired, and PHIA was slightly reduced. Marked lethargy, agitation, and a 20% mortality were present during this gram-positive infection. Results of this study support the concept that an impairment of lipid disposal mechanisms, particularly during gmmnegative sepsis with S. typhimurium, may significantly contribute to the observed hypertriglyceridemia.

A

LTERED PLASMA lipid levels have been observed during a variety of infectious illnesses in both human and animal studies.lm6 Elevation of plasma total lipid and triglyceride values have been reported in patients during gram-negative septicemia, viral hepatitis, and the convalescent phase of some viral illnesses but not during gram-positive septicemia or influenza in humans.‘-’ Patients with severe gram-positive infection have demonstrated decreased free and esterified cholesterol levels as the major plasma lipid alteration.’ Experimental infection in rhesus monkeys has shown variable increases in plasma triglycerides during Salmonella typhimurium and Diplococcus pneumoniae sepsis with decreased cholesterol and variable free fatty acid levels.4 While the elevation of plasma lipids has been quite well established in certain infectious states, mechanisms responsible for this response are poorly understood.’ It has been suggested that increased free fatty acid fluxes observed

From the U. S. Army Medical Research Institute of Infectious Diseases, Frederick. &Id. Received for publication March 26. 1975. In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care,” as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. The facilities are fully accredited by the American Association of Accreditation of Laboratory Animal Care. Presented in part before the Federation of Ametican Societies for Experimental Biology, Atlantic City, N.J.. ApriltJ. 1974. Reprint requests should be addressed to Major Robert L. Kaufmann. Physical Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Md. 2I 701. 0 1976 by Grune & Stratton, Inc. Metabolism, Vol. 25, No. 6 (June), 7976

615

KAUFMANN

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ET AL.

by some investigators would promote hepatic lipogenesis.3,4 The observations that elevated triglycerides appear in the form of very low density lipoproteins (VLDL) formed predominately in the liver during convalescence after viral infection in humans’ or as prominent pre-beta bands on lipoprotein electrophoresis during S. typhimurium sepsis in rhesus monkeys4 support this conclusion. This has led to an assumption that increased hepatic lipid synthesis could be the major cause of elevated plasma triglycerides during infection: this assumption is consistent with fatty infiltration of the liver observed during infectious disease or after intravenous endotoxin.8,9 However, during an infection which produces anorexia one would expect lipoprotein lipase activity to be reduced since detectable in vivo activity of this lipid-clearing enzyme is decreased by fasting alone.” Likewise, in a steady-state equilibrium, endogenously produced triglycerides must be continuously broken down by lipoprotein lipase to prevent an accumulation of triglyceride in plasma. In this study lipid disposal mechanisms were assessed by oral lipid loading, intravenous lipid loading’ and postheparin lipolytic activity tests measured in separate groups of male rhesus monkeys immediately before and during experimental S. typhimurium and D. pneumoniae sepsis. Results of these tests support the contention that lipid disposal mechanisms are impaired particularly during gram-negative sepsis and may significantly contribute to the observed elevation in serum triglyceride concentrations. MATERIALS

AND METHODS

All studies were performed with 2.3-4.3 kg, male, rhesus monkeys (Macacu mtrlntta) which had been observed and maintained on a commercial diet (Wayne Monkey Diet, Allied Mills, Inc., Chicago, Ill.) and fruit for a minimum of 4 mo before study. Monkeys were placed in individual restraint chairs with free access to food and water 5-6 days before studies were performed. Studies were initiated by 9:00 a.m. so that overnight fasting (approximately I2 hr) samples could be obtained. After the l2-hr fast, saphenous veins were cannulated with l6-gauge intravenous catheters and samples were obtained using a double syringe technique. Baseline lipid disposal studies were performed. Monkeys were than intravenously inoculated with either IO9 S. typhimurium. IO* D. pneumoniae. or with 1.0 ml physiologic saline for controls. All monkeys were studied both before and 48 hr after inoculation, a time when each type of bacterial illness was uniformly severe and fasting serum triglyceride elevations were greatest. Complete blood counts and blood cultures were obtained before and 48 hr after inoculation to confirm the presence and nature of the infected state. The ability to dispose of triglycerides after oral fat loading was measured by giving 4 g/kg of a corn oil emulsion, Lipomul (Upjohn Company, Kalamazoo, Mich.), containing 0.6 mg sodium saccharine/g lipid by nasogastric tube. Intravenous lipid tolerance was tested in other groups of control and infected monkeys by infusing 0.5 g/kg of a soybean-oil emulsion. lntralipid (Cutter Laboratories, Berkeley, Calif.), over 30 min. Sequential serum samples were analyzed for triglycerides, total cholesterol, and free fatty acids by automated techniques.“.” When no differences were found between results of the first or preinoculation test and the pre- and postsaline tests of controls, results were combined as control values unless stated otherwise. Total postheparin plasma lipolytic activity was assessed in other control and infected groups by measuring the lipolytic activity before (baseline) and at I, 2, 6, IO, and I6 min after 0.1 mg/kg intravenous sodium heparin (Lipo-Hepin, Riker Laboratories, Inc., Northridge, Calif.). Blood samples were immediately placed in ice and plasmas were then quickly separated, frozen in liquid nitrogen, and stored at -70” until analyses were completed. The in vitro lipase assay was completed following the method of Fredrickson, Ono, and Davis.13 Individual glass stoppered tubes each containing 0.5 ml of 100 mg albumin (bovine fraction V, Armour Pharmaceutical Co., Chicago, Ill.) in 0.1 M ammonium sulfate buffer pH 8.6, 0.3 ml lntralipid (15 mg) and 0.2 ml

LIPID

DISPOSAL

DURING

617

SEPSIS

plasma with a total incubation volume of 1 .O ml were placed in a Dubnoff shaker at 37”. Duplicate tubes were removed at 5 min as a zero time blank and then at 15, 30, and 45 min. The reaction was stopped immediately by adding 6 ml chloroform and 3 ml 0.1 M NaPO., buffer pH 6.2 for free fatty acid extraction. A linear production of unesterified fatty acid during 45 min of incubation was required for an adequate enzyme assay, a condition fulfilled in 95% of all assay samples. Nonlinear assays were excluded from calculations. Samples obtained 48 hr after saline inoculation in controls and 48 hr after infection were assayed at the same time. Enzyme activity remained constant for up to a month when samples were stored at -7o’C and most analyses were completed within 2 wk. Postheparin lipolytic activity (PHLA) was determined at each time interval. In addition, to minimize the possible effect of any variation in peak times, the area over baseline for each study was also calculated by summation of areas under the curve from I to I6 min. PHLA studies were performed after I2 hr fasting in control monkeys and also after I2 hr fasting (48 hr after inoculation) in bacterially infected monkeys. RESULTS

Following inoculation with S. typhimurium, monkeys displayed slight lethargy, occasional diarrhea, and fever. Blood cultures became positive and increased polymorphnuclear leukocyte counts were observed without an increase in total leukocytes. Clinical illness was observed uniformly at 48 hr postinoculation in S. typhimurium-infected monkeys but spontaneous recovery generally occurred. These monkeys rarely appeared to be severely ill and the group experienced only 2% overall mortality. In contrast, monkeys inoculated with D. pneumoniae consistently showed marked lethargy, agitation, anorexia, and fever at 48 hr. These monkeys had positive blood cultures and prominent polymorphonuclear leukocytosis. The greater degree of clinical illness correlated with a higher mortality of 20% in this group. After exhibiting moderately severe illness 48 hr postinoculation, D. pneumoniae-infected monkeys either became more severely ill and died or underwent spontaneous recovery. Table 1 shows fasting serum triglyceride, cholesterol, and free fatty acid (FFA) concentrations 48 hr after inoculation with either S. typhimurium or D. pneumoniae. Triglyceride values are significantly higher in S. typhimuriuminfected monkeys when compared with either D. pneumoniae-infected (p < 0.001) or control (p < 0.001) monkeys. While cholesterol levels were lowest in the D. pneumoniae-infected monkeys, these measurements were not significantly different among the three groups. FFA levels were the lowest in the S. typhimurium-infected group and were significantly lower than the D. pneumoniaeinfected monkeys (p < 0.01). The effect on triglyceride values of either oral Lipomul administration (4 g/kg) or continued fasting in noninfected normal monkeys is shown in Fig. 1. This amount of lipid emulsion is normally sufficient to elevate triglyceride Fasting Serum Triglyceride,

Table I.

Cholesterol,

and Free Fatty Acid Concentmtions*

Rhesus Monkevs 48 hr After Inoculation With Either 5. tvdtimurium N

Group Controls St.-infected La-infected *Means t

f

monkeys monkevs

Triglyceride

Cholesterol

mg/dl

120+

mg/dl 9

Free Fatty Acids@Eq/L

19

31*

3

749

f

58

41

149+

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649

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35

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7

114zts

811

f

34

S.E.M.

Higher than either control

in

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or D. pneumonioe-infected

$Lowerthan D. pneumoniae-infected

monkeys,

monkeys,

p < 0.01.

p < 0.001.

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Fig. 1. Triglyceride values (means f SEM) observed in noninfected monkeys ofter 4 g/kg oral fat loading compared to fasting controls.

values slightly, but significantly, during a 4- to 24-hr period following its administration. A peak value of 45.8 mg/dl occurred at 20 hr. Figure 2 uses a different scale to show the triglyceride response after oral Lipomul administration in infected and control monkeys. S. typhimuriuminfected monkeys had the highest triglyceride levels with values in excess of 550 mg/dl by 24 hr. Increased triglyceride concentrations were also observed in D. pneumoniue-infected monkeys with values consistently lower than S.

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Triglyceride valuer (means f SEM) in monkeys during 24 hr ofter oral lipid loading Fig. 2. (4 g/kg) beginning 48 hr oher inoculation with 5. typhimurium /St) 01 D. pneumonias (0~).

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HOURS AFTER ORAL LIPID Free fatty acids (means f SEM) in monkeys during 24 hr after oral lipid loading fig. 3. (4 g/kg) beginning 48 hr after inoculation with 5. typhimurium (St) or D. pneumoniae (Dpj.

typhimurium-infected monkeys but higher than controls. A further analysis of these data with respect to the rate of increase in triglyceride levels showed that between 2 and 12 hr the rate of rise (or the slope) in the S. typhimurium group was twice as great as that measured in the D. pneumoniae-infected monkeys. This difference was significant when either individual monkey data were calculated (p < 0.05) or when the group mean values were used (p < 0.01). FFA values obtained after oral Lipomul loading are shown in Fig. 3. While LIPID (o.sgmlhg)

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MINUTES Free fatty acids (means f SEM) in monkeys during intmvenous lipid loading (0.5 g/kg) 46 hr after inoculotion with 5. typhimurium (N = 12), D. pneumoniaa (N = 12), or soline (N = 8).

no FFA differences were observed between S. typhimurium-infected and control monkeys, FFA were significantly lower in D. pneumoniae-infected monkeys compared to controls during the period from 4 to 24 hr after giving Lipomul. Results of intravenous lipid loading with Intralipid in both control and infected monkeys are shown in Fig. 4. Peak triglyceride concentrations were observed at the end of the lipid infusion at which time no difference was observed between control and either group of infected monkeys. S. fyphimurium-infected monkeys had significantly (p < 0.01) higher triglyceride concentrations at 120, 180, and 240 min after initiation of the infusion compared to controls and were also higher than D. pneumoniue-infected monkeys at 180 and 240 min. No significant difference was observed in lipid tolerance between D. pneumoniaeinfected and control monkeys. Free fatty acid responses during intravenous lipid loading are displayed in Fig. 5. Control monkeys showed significantly higher free fatty acids at 30, 45, 60, and 90 min after intravenous Intralipid. Lower free fatty acid levels were observed after both infections with no difference between S. typhimurium and D. pneumoniae-infected monkeys. An analysis of lipolytic enzyme activity as measured by postheparin lipolytic activity (PHLA) is presented in Table 2. While preinoculation and postinoculation studies were performed in control and infected monkeys, PHLA during the second study in controls were slightly, but not statistically lower compared with the first test. Nevertheless, only the values for the second test in controls were used to compare with the second or postinoculation study in infected monkeys. Normal monkeys demonstrated highest PHLA values at 1 or 2 min after heparin with activities gradually decreasing thereafter. In D. pneumoniaeinfected monkeys, PHLA was significantly lower than control monkeys at 2 min and higher than S. typhimurium-infected monkeys at 1 and 2 min postheparin.

LIPID

DISPOSAL

Table 2.

DURING

SEPSIS

621

Fosthepatin lipolytic Activity After 0.1 mg/kg Heparin in Control Monkeys and 48 hr

After Monkeys Were Inoculated With Either 0. pneumonias or 5. typhimurium Organisms Porthcporin N

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Controls

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0.016

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< 0.001.

tt p < 0.01. min (mean

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However, the pattern of response was similar to normal with values gradually decreasing after 2 to 16 min. The pattern of response was different in S. typhimurium-infected monkeys with peak activity delayed to 10 min postheparin. S. ryphimurium-infected monkeys demonstrated significantly lower values at 1,2, and 6 min compared to controls with the overall area also decreased compared to control monkeys. DISCUSSION

This study demonstrates the close association of elevated serum triglyceride values and impaired lipid disposal mechanisms during two model experimental bacterial infections in the rhesus monkey. While previous studies have measured serum lipid changes during a variety of infectious diseases,im6 little attention has been directed toward rates of triglyceride synthesis or degradation. Because uniform illness and maxima1 fasting serum triglyceride concentrations were present 48 hr postinoculation in each model infection under study, we selected this time period to investigate lipid disposal mechanisms. Although the pneumococcal infection was clinically more severe, the lipid abnormalities were of greater magnitude in monkeys with salmonellosis. This difference between the infections in monkeys is consistent with clinical observations in man7 which suggest that lipid abnormalities are most common and most severe in patients infected with gram-negative bacteria. Elevated fasting triglyceride values have previously been reported during gram-negative sepsis in both human and animal studies.‘+4*‘2 In the present study hypertriglyceridemia was uniformly observed 48 hr after inoculation with S. typhimurium and occurred in the presence of a marked impairment of lipid disposal mechanisms. In contrast, during gram-positive sepsis produced by D. pneumoniue. mean triglyceride concentrations were not significantly elevated when compared to control values and minimal evidence of impaired lipid disposal was observed despite more profound morbidity and greater mortality. However, hypertriglyceridemia was observed in severely infected D. pneumoniae-infected monkeys shortly before death at which time tests of lipid disposal were not performed. These results are similar to those reported by Fiser et al. and emphasize the importance of severity of illness when evaluating lipid values during gram-positive sepsis4

622

KAUFMANN

ET AL.

While lipid absorption during infection has been found to be either slightly decreased or unaffectedL4,‘5 little information is available on the removal of absorbed lipid during infectious illness. Monkeys were initially tested for their tolerance to an exogenous oral lipid load using Lipomul, a corn oil emulsion. While slight but significant differences were observed between triglyceride concentrations with and without Lipomul administration in noninfected monkeys, differences in the infected state were marked. These results suggest that absorption was either not significantly impaired or was possibly increased during infection. At the same time it would appear that the removal of absorbed triglyceride from plasma was impaired resulting in a greater accumulation of triglyceride during S. typhimurium than during D. pneumoniae sepsis. The increased fasting triglyceride levels in S. typhimurium-infected monkeys before giving Lipomul undoubtedly contributed to the triglyceride measurements after lipid loading. Nevertheless, the increment of triglyceride from hours 2 to 12 after correction for baseline values, was significantly greater in S. typhimuriuminfected monkeys than in the D. pneumoniae group. Additional studies employing intravenous Intralipid helped to resolve the problems of variability in lipid absorption as well as the effect of elevated triglyceride concentrations on subsequent lipid clearance. Impairment of lipid clearance was clearly demonstrated by higher concentrations of serum triglycerides from 120 to 240 min in S. typhimurium-infected monkeys. While D. pneumoniae-infected monkeys had impaired clearance of oral lipid, they cleared intravenous lipid without difficulty. The reason for this is unclear but demonstrates the necessity to further study lipid absorption during infection. An analysis of lipolytic enzyme activity as reflected by PHLA showed reduced activity in both bacterial infections with a much greater effect observed during S. typhimurium sepsis. The decreased PHLA in S. typhimurium infected monkeys can not be attributed to fever, relative starvation or increased stress since the D. pneumoniae-infected monkeys had significantly greater illness with less impairment of PHLA. Likewise, it is unlikely that increased lipid pools have interfered with this enzyme activity since increased PHLA has been shown to correlate positively with increased triglycerides in some cases of hypertriglyceridemia16 and activity is not decreased in usual clinical cases of endogenous I7 Recent studies in humans suggest that postheparin hypertriglyceridemia. plasma contains predominantly hepatic triglyceride lipase (46x-95%) as well as lipoprotein lipase (approximately 30%) and phospholipase A, (approximately 8%);‘e*‘9 these results were obtained under different assay conditions and the distinction between hepatic and extrahepatic lipases requires further distinction. Likewise, it is not possible to determine from our present studies which lipase activities have been decreased and further studies will be required to clarify this point. However, the failure to clear exogenous lipid would suggest reduced lipoprotein lipase activity. In contrast to some instances of hyperthyroidism and starvation when reduced PHLA is associated with normal triglyceride values,20 decreases in total postheparin lipolytic activity and impaired disposal of exogenous lipid were consistently observed in the presence of hypertriglyceridemia in our monkeys with salmonellosis. Significantly altered cholesterol levels were not observed during these studies.

LIPID DISPOSAL

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While levels in D. pneumoniue-infected monkeys were consistently lower than in those infected with S. typhimurium, this difference was not statistically significant. Lower cholesterol levels have been previously reported during both bacteria13p4 and viral illnesses’ and may reflect the generalized catabolic state during infection.14 Fasting free fatty acids (FFA) measured during S. typhimurium sepsis in this study were not higher than controls. If increased serum triglyceride concentrations were predominately due to increased FFA fluxes from adipose tissue,’ we might expect to observe increased FFA levels even though FFA fluxes were not measured. The lower FFA levels in D. pneumoniae-infected monkeys after both oral and intravenous lipid loading without evidence of a major defect in total postheparin lipolytic activity would support the concept of a greater metabolic utilization of FFA in these sicker animals compared with S. typhimurium-infected monkeys.’ The association of elevated fasting triglyceride values, intolerance to exogenous lipid loading, and decreased PHLA suggest that lipid disposal is truly impaired during bacterial sepsis. Likewise we propose that the defective lipid disposal observed in this study either produced or contributed to the elevated triglyceride values observed during gram-negative bacterial sepsis. ACKNOWLEDGMENT The authors gratefully acknowledge the assistance of Miss Karen SP4 Gilbert0 Vega, SP4 Ronald Bynum, and SP5 George Johnson.

Bostian,

Mr. Billy Blackburn,

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9. Hirsch RL, McKay DG, Travers RI, Skraly RK: Hyperlipidemia, fatty liver, and bromsulfophthalein retention in rabbits injected with bacterial endotoxins. J intravenously Lipid Res 5:563-568, 1964 10. Arons DL, Schreibman PH, Arky RA: Post-heparin lipolytic and monoglyceridase activities in fasted man. Proc Sot Exp Biol Med 137:780-782, 1971 11. Rush RL, Leon L, Turrell J: Automated simultaneous cholesterol and triglyceride determination on the AutoAnalyzer II instrument. Advances in Automated Analysis: Technicon International Congress 1:503-507, 1970 12. Dalton C, Kowalski C: Automated calorimetric determination of free fatty acids in biologic fluids. Clin Chem 13:744751, 1967 13. Fredrickson DS, Ono K, Davis LL: Lipolytic activity of postheparin plasma in hyperglyceridemia. J Lipid Res 424-33, 1963 14. Beisel WR: Interrelated changes in host metabolism during generalized infectious illness. Am J Clin Nutr 25:1254-1260, 1972 IS. Du Bois EF: The absorption of food in typhoid fever. Arch Intern Med l&177-195, 1912 16. Sailer S, Sandhofer F, Braunsteiner H:

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Regulation of endogenous lipoprotein-lipase activity in the plasma of normal subjects and patients with essential hyperlipaemia. German Med Mon 11:4l-43, 1966 17. Fredrickson DS, Levy RI, Lees RS: Fat transport in lipoproteins-An integrated approach to mechanisms and disorders. N Engl J Med 276:215-224,273-281,1967 18. Krauss RM, Levy RI, Fredrickson DS: Selective measurement of two lipase activities in postheparin plasma from normal subjects

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ET Al.

and patients with hyperlipoproteinemia. J Clin Invest 5411107-I 124, 1974 19. Ehnholm C, Huttunen JK, Kinnuen PJ, Miettinen TA, Nikkill EA: Effect of oxandrolone treatment on the activity of lipoprotein lipase, hepatic lipase and phospholipase A of human postheparin plasma. N Engl J Med 292: 1314-1317.1975 20. Arons DL, Screibman PH. Downs P, Braverman LE. Arky RA: Decreased postheparin lipases in Grave’s Diseases. N Engl J Med 2861233-237, 1972