Int. J. Med. Sci. 2010, 7 http://www.medsci.org 101 I I n n t t e e r r n n a a t t i i o o n n a a l l J J o o u u r r n n a a l l o o f f M M e e d d i i c c a a l l S S c c i i e e n n c c e e s s 2010; 7(3):101-109 © Ivyspring International Publisher. All rights reserved Research Paper Growth of Microorganisms in Total Parenteral Nutrition Solutions Con- taining Lipid Takashi Kuwahara 1 , Kazuyuki Shimono 1 , Shinya Kaneda 1 , Takumi Tamura 1 , Masao Ichihara 2 , Yoshifumi Nakashima 1 1. Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan. 2. Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan. Corresponding author: Takashi Kuwahara, Ph.D., Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., 115 Tateiwa, Naruto, Tokushima 772-8601, Japan. Telephone: +81 88 685 1151 (Ext. 678) Fax: +81 88 684 0553; E-mail: kuwahat2@otsukakj.co.jp Received: 2010.02.01; Accepted: 2010.05.17; Published: 2010.05.18 Abstract Background: To identify the microorganisms that can grow rapidly in total parenteral nu- trition (TPN) solutions, we investigated the growth of the major causes of catheter-related blood stream infection (Staphylococcus aureus, Serratia marcescens, Bacillus cereus, and Candida albicans) in TPN solutions containing lipid. Methods: The pH value of a TPN solution con- taining lipid (pH 6.0, containing 20 ppm of NaHSO 3 ) was adjusted by the addition of HCl to 5.7, 5.4, or 4.9. The pH value of another TPN solution (pH5.5, containing 400 ppm of NaHSO 3 ) was adjusted by the addition of NaOH to 5.9, 6.3, or 6.8. A specific number of each microorganism was added to 10 mL of each test solution and incubated at room temperature. The number of microorganisms was counted as colony forming units at 0, 24, and 48 hrs later. Results: C albicans increased similarly at any pH values in the TPN solution. The bacterial species also increased rapidly at pH6.0 in the solution containing 20 ppm of NaHSO 3 , but growth was suppressed as the pH value was reduced, with growth halted at pH4.9. However, these bacterial species did not increase, even at pH5.9, in the other solution containing 400 ppm of NaHSO 3 . Conclusions: These results suggest that Candida species can grow rapidly in almost all TPN solutions regardless of the acidity, lipid, and NaHSO 3 ; also, some bacterial species may grow in TPN solutions containing lipid unless the pH value is 5.0 or less. Therefore, each TPN solution should be examined whether or not the bacterial species can proliferate. Key words: CRBSI, microbial growth, TPN solution, lipid, pH, bisulfite INTRODUCTION Catheter-related blood stream infection (CRBSI) is one of the most common complications of intra- venous catheters. 1-3 To reduce or prevent CRBSI, fac- tors that enhance or inhibit microbial growth in pa- renteral solutions should be investigated and identi- fied. Total parenteral nutrition (TPN) solutions are considered to be relatively good growth media for microorganisms, 1,4 whereas a number of investigators have shown that TPN solutions are poor growth me- dia for most microorganisms that cause CRBSI, with the exception of Candida species. 5-9 Likewise, we have previously confirmed that while bacterial species do not grow in TPN solutions without lipid, Candida species grow rapidly. 10 Our data have shown that bacterial species cannot grow due to the acidity, but Candida species can grow regardless of the acidity. 10 In contrast, most microorganisms grow rapidly in com- mercial lipid emulsions, 11-15 and sporadic CRBSI and Int. J. Med. Sci. 2010, 7 http://www.medsci.org 102 outbreaks have been traced to contaminated lipid emulsion. 16,17 Because lipid emulsions have been shown to contribute to the rapid growth of most mi- croorganisms that cause CRBSI, it can be assumed that TPN solutions containing lipid promote the microbial growth compared with TPN solutions without lipid. 18 Consequently, the Center for Disease Control and Prevention (CDC) has recommended that when any infusion solutions containing lipid are administered, the entire delivery system such as the administration set be replaced every 24 hours. 19 As the major causes of CRBSI, Staphylococcus aureus, Staphylococcus epidermidis, Serratia mar- cescens, Escherichia coli, Klebsiella pneumonia, Can- dida albicans, etc. were shown. 2,3,6,11 Furthermore, blood stream infection outbreaks of Bacillus cereus via intravenous line were recently reported in Japan. 20 In the present study, we investigated the growth in TPN solutions containing lipid of the same microorganisms studied previously: 10 ie, Staphylococcus aureus as a delegate of gram positive cocci, Serratia marcescens as a delegate of gram negative rods, Bacillus cereus as a delegate of gram positive rods, and Candida albicans as a delegate of fungi. To clarify whether the acidity of TPN solutions suppresses microbial growth in the presence of lipid, the growth of all strains employed were investigated at various pH values in a commer- cial TPN solution containing lipid in the first experi- ment. To identify which factors enhance or inhibit the growth of each microorganism, we supplemented a TPN solution containing lipid with multivitamins, supplemented a TPN solution that did not contain lipid with lipid, and increased the bisulfite concentra- tion in a TPN solution containing both lipid and mul- tivitamins in the following experiments. MATERIALS AND METHODS Microorganisms employed A standard American Type Culture Collection (ATCC) strain and 1 clinical isolate were used for each microorganism; the standard strain ATCC6538 and the clinical isolate N3 of Staphylococcus aureus, the standard strain ATCC13880 and the clinical isolate N4 of Serratia marcescens, the standard strain ATCC11778 and the clinical isolate H2 of Bacillus cereus, and the standard strain ATCC10231 and the clinical isolate N7 of Candida albicans. Test solutions A commercial TPN solution containing lipid (ML; MIXID-L, Otsuka Pharmaceutical Factory, Inc., Japan), a commercial TPN solution without lipid (AT1; AMINOTORIPA-1, Otsuka Pharmaceutical Factory, Inc.), multivitamins (MV; Otsuka MV Injec- tion, Otsuka Pharmaceutical Factory, Inc.), a 20% lipid emulsion (IL; Intralipos 20%, Otsuka Pharmaceutical Factory, Inc.), and a physiological saline were used. The compositions of ML and AT1 are shown in Table 1. Table 1. The compositions of ML and AT1 Solution ML AT1 ( volume ) 900 mL 850 mL Amino acids 30.0 g 25.0 g Glucose 110.0 g 79.8 g Fructose - 40.2 g X y litol - 19.8 g Li p id 15.6 g - Na + 35 mE q 35 mE q K + 27 mE q 22 mE q M g 2+ 5 mE q 4 mE q Ca 2+ 8.5 mE q 4 mE q Cl － 44 mE q 35 mE q SO 4 2 － 5 mE q 4 mE q Acetate － 25 mE q 44 mE q Gluconate － 8.5 mE q 4 mE q Citrate 3 － - 10 mE q P 150 m g 154 m g Zn 10 μ mol 8 μ mol NaHSO 3 17 m g 412 m g p H 6.0 5.5 Osmotic p ressure ratio to p h y siolo g ical saline A pp rox. 4 A pp rox. 5 Int. J. Med. S c Experi m The pH val u saline (OPR , molalit y of p NaHSO 3 of ppm (about lutions of M addition of 0 Experi m supplement e Experi m and used as OPR, and co n approximat e spectively. T justed to 5.9 , NaOH. Eac h 1/10 volum e (S+AT1V). Experi m and used as solutions of the addition and MLV( p (MLV+100 p ppm). Addition o f sampling A spec i was added t plastic tube s room tempe r tion was sa m dition of mi c Measurem e Each a l oculated in a in duplicate. tion was dil u saline befor e at 37ºC, the n each microo r mean CFU o aliquot, and was calculat e aliquot vol u shown as v graphs. As o growth, 5,12,13 , not anal y ze d ance of the s sessable wit h c i. 2010, 7 m ent 1: ML w u e, osmotic p r , the osmolal p h y siolo g ical ML were 6. 0 20 ppm), re s M L were ad j u s 0 .5 mol/L H C m ent 2: ML(p H e d with MV ( M m ent 3: AT1 w the base sol u n centration o e l y 5, and 41 2 T he other pH , 6.3, or 6.8 b h pH solutio n e of IL (L+A T m ent 4: ML w the base so l MLV were a of 0.5 mol/ L p H5.4) wer e p pm) or 200 p f microorga n i fic number t o each 10 m s , and all tub e r ature (23-26 º m pled at 0, 2 4 c roor g anisms . e nt of viable l iquot of tes t a So y bean Ca When neces s u ted 10-fold t o e inoculation . n umber of c o rg anism was o f duplicate d number of e e d b y usin g t h u me, and dil u v alues of C F o ther experi m , 21,22 the data d statisticall y, s e kinds of d h out statistic a w as used as t h r essure ratio t it y of each s saline), and c 0 , approxim a s pectivel y . T h s ted to 5.7, 5. C l. H 6.0), ML(p H M LV) were u w as supple m u tion (AT1V) . o f NaHSO 3 of 2 ppm (abou solutions of by the additi o n of AT1V w T 1V) or ph ys w as supplem e l ution (MLV) d j usted to 5. 7 L HCl. Besid e e added w p pm of Na H n ism, incub a of each test m L of test sol u e s were allo w º C). An aliq u 4 , and 48 ho u . microorga n t solution s a sein Di g est ( S s ar y , the aliq u o 10 7 -fold wi t . After a 24- h o lon y formin g counted for d ata was cal c e ach microor g h e number o f u tin g ratio. T F U/mL in s e m ental studi e obtained in t , because bi o d ata is consi d a l anal y sis. h e base soluti o t o ph y siolo g i c olution/the o c oncentratio n a tel y 4, and h e other pH s 4, or 4.9 b y t H 4.9), and th o u sed. m ented with M . The pH val u AT1V were 5 t 400 ppm), r AT1V were a o n of 0.5 mol / w as added w i s iolo g ical sal i e nted with M . The other p 7 , 5.4, or 5.0 e s, MLV(pH 5 w ith 100 p p H SO 3 (MLV+ 2 a tion and microor g ani s u tions in ste r w ed to stand u ot of test so l u rs after the a n isms a mpled was i S CD) a g ar pl a u ot of test so l t h ph y siolo g i c h our incubat i g units (CFU) each plate. T c ulated for e a g anism per m f CFU per pl a T he results a e mi-lo g arith m e s of micro b t his stud y w e o lo g ical si g ni f d ered to be a o n. c al o s- n of 17 s o- t he o se M V u e, 5 .5, r e- a d- / L i th i ne M V p H by 5 .7) p m 2 00 s m r ile at l u- a d- i n- a te l u- c al i on of T he a ch m L a te, a re m ic b ial e re f ic- a s- RES U Expe r S 6.0, b u reduc e marces c increa s not in c cereus presse pH of cans i n (Fi g ur e Figur e aureus adjuste Figur e in ML ( p by addi U LTS r iment 1 . aureus incr e u t g rowth wa s e d, comin g t o c ens increase d s ed but with s c rease at pH increased at d as the pH v 4.9 (Fi g ure 3 ) n creased rapi d e 4). e 1. Effect of in ML (pH6.0; d by addition o e 2. Effect of p H p H6.0; NaHS O tion of 0.5 m o e ased in ML s suppresse d o a halt at a p d rapidly in M s uppression a values of 5.4 a pH of 6.0. v alue was re d ) . However, b d l y and simi l pH on the g r NaHSO 3 , 20 p o f 0.5 mol/L H H on the grow t O 3 , 20 ppm). T h o l/L HCl. http://www. m at the ori g in d as the pH v a H of 4.9 (Fi gu M L at a pH o f a t a pH of 5.7 , and 4.9 (Fi gu Its g rowth w d uced, decre a b oth strains o l arl y at an y p r owth of Stap h p pm). The pH Cl. t h of Serratia m h e pH value wa s m edsci.org 103 al pH of a lue was u re 1). S. f 6.0, also , and did u re 2). B. w as sup- a sin g at a o f C. albi- p H value h ylococcus value was m arcescens s adjusted Int. J. Med. S c Figure 3. Eff e (pH6.0; NaH S addition of 0. 5 Figure 4. Eff e ML (pH6.0; N by addition o f Experimen t Supple m the g rowth resume the cerning S. m a not affect th e c i. 2010, 7 e ct of pH on th S O 3 , 20 ppm). 5 mol/L HCl. e ct of pH on t h N aHSO 3 , 20 pp f 0.5 mol/L H C t 2 m entation wi of S. aureus g rowth at a a rcescens, B. c e e m both at p H e growth of B a The pH value h e growth of C m). The pH v a C l. th MV to M L at a pH of 6 pH of 4.9 ( F e reus and C. a H 6.0 and at p H a cillus cereus in M was adjusted C andida albican s a lue was adjus t L enhanced o n 6 .0 but did n F i g ure 5). C o a lbicans, MV d H 4.9 (Fi g ure M L by s in t ed n l y n ot o n- d id 5). Figur e cescens , pH4.9 t amins ) Expe r T L+AT 1 diluti n tion. I n marces c well a s at pH 6-8), s i S u B. cere u 5.9, d e result s 6.3 an d volu m g rowt h A e 5. Growth o , Bacillus cere u in ML and in M ) . r iment 3 T he OPR of 1 V (or S+AT1 ng effect fro m n AT1V, the c ens and B. c e s at the ori g i n 6.3 and inc r i milar to the r u rprisin g l y , n u s increased i e spite the pr e s conflicted w d pH 6.8, ho w m e of IL to AT 1 h of these ba c A ddition of 1 / o f Staphylococ c u s, and Candid a M LV (ML sup p AT1V was 4 V) was 4.3 (o m the additio n standard st r e reus did not n al pH of 5.5 r eased rapid l r esults of our n one of S. au r i n L+AT1V a e sence of lipi d w ith those of w ever, suppl e 1 V (L+AT1V ) c terial specie s / 10 volume o http://www. m c us aureus, Ser a albicans at p p lemented wit 4 .6, and the r 4.2) as a res u n of the isoto r ains of S. a increase at p but increase ly at pH 6.8 previous stu d r eus, S. marce s a t either pH 5 d (Fi g ures 6- 8 Experiment 1 e mentation w ) enhanced m s (Fi g ures 6-8 ) o f ph y siolo g i c m edsci.org 104 ratia mar- p H6.0 and h multivi- OPR of u lt of the nic solu- a ureus, S. p H 5.9 as d slowly (Fi g ures dy . 10 s cens and 5 .5 or pH 8 ). These 1 . At pH w ith 1/10 m ildl y the ) . c al saline Int. J. Med. S c to AT1V (S + OPR from 4 bacteria less IL (Fi g ures 6 Experimen t The g r o halted only a ppm of Na H S. aureus. H o added, the gr pH 5.7 and (Figure 9). Figure 6. Eff e adjusted by a d saline (S+AT1 Figure 7. Ef f adjusted by a d saline (S+AT1 c i. 2010, 7 + AT1V) cont r 4 .6 to 4.2, e n sli g htl y tha n -8). t 4 o wths of 2 str a a t pH 5.0 (Fi g H SO 3 was not e o wever, whe n r owth of the s the clinical i s e ct of lipid on d dition of 0.5 m V). f ect of lipid o n d dition of 0.5 m V). r ibuted to t h n hancin g the n did supple m a ins of S. aure u g ure 9). The e nou g h to st o n 200 ppm o s tandard stra i s olate was h a the growth o m ol/L NaOH. T n the growth o m ol/L NaOH. T h e reduction g rowth of t m entation w i u s in MLV w e addition of 1 o p the g rowt h o f NaHSO 3 w i n was halte d a lted at pH 5 o f Staphylococc u T o AT1V was a o f Serratia ma r T o AT1V was a of t he i th e re 1 00 h of w as d at 5 .4 T were h pondi n tion o f strain s T MLV w g rowt h (Fi g ur e halted and t h g rowt h u s aureus in A T dded 1/10 vol u r cescens in AT dded 1/10 vol u T he growth o f h alted at pH 5 ng with the r f 200 ppm of N s of S. marcesc e T he growth o f w as halted b o h of the clin e 11). The a the g rowth o h e addition o h of the clini c T 1V (pH5.5; N u me of 20% lip i 1V (pH5.5; N a u me of 20% lip i f 2 strains of 5 .0 and pH 5 r esults of Ex p N aHSO 3 halt e e ns at pH 5.7 f the standar d o th at pH 5. 0 ical isolate w a ddition of 2 o f both strain s o f 100 ppm o c al isolate at p N aHSO 3 , 400 p i d emulsion (L + a HSO 3 , 400 p p i d emulsion (L + http://www. m S. marcescen s 5 .4 (Fi g ure 10 ) p eriment 1. T e d the g rowt h (Fi g ure 10). d strain of B. 0 and pH 5.4 , w as halted a t 2 00 ppm of s of B. cereus a o f NaHSO 3 h a p H 5.4 (Fi g ur e pm). The pH v + AT1V) or ph y p m). The pH v + AT1V) or ph y m edsci.org 105 s in MLV ) , corres- T he addi- h of both cereus in , and the t pH 5.0 NaHSO 3 a t pH 5.7, a lted the e 11). v alue was y siological v alue was y siological Int. J. Med. S c Figure 8. Eff e addition of 0. (S+AT1V). Figure 9. Eff e tamins). The p (+100ppm or c i. 2010, 7 e ct of lipid on t 5 mol/L NaO H e ct of bisulfite p H value was a +200ppm). t he growth of B H . To AT1V w concentratio n a djusted by a d B acillus cereus i n w as added 1/1 0 n on the grow t d dition of 0.5 m n AT1V (pH5. 5 0 volume of 2 t h of Staphyloc o m ol/L HCl. T o 5 ; NaHSO 3 , 4 0 0% lipid emul s o ccus aureus in o MLV was ad d 0 0 ppm). The p s ion (L+AT1V ) MLV (ML sup d ed 100 ppm o http://www. m H value was a d ) or physiolog plemented wi t o r 200 ppm o f m edsci.org 106 d justed by ical saline t h multivi- f NaHSO 3 Int. J. Med. S c Figure 11. E f The pH value +200ppm). c i. 2010, 7 f fect of bisulfit e was adjusted b e concentratio b y addition of 0 n on the gro w 0 .5 mol/L HCl. Figure 10. E Serratia marc e The pH value w as added 1 +200ppm). w th of Bacillus c e To MLV was a E ffect of bisul f e scens in MLV ( w as adjusted 00 ppm or 2 e reus in MLV ( M dded 100 ppm f ite concentr a ML suppleme n by addition of 2 00 ppm of N M L suppleme n or 200 ppm o http://www. m a tion on the g n ted with multi 0.5 mol/L HC l N aHSO 3 (+1 0 n ted with multi f NaHSO 3 (+1 0 m edsci.org 107 g rowth of vitamins). l . To MLV 0 0ppm or vitamins). 0 0ppm or Int. J. Med. Sci. 2010, 7 http://www.medsci.org 108 DISCUSSION To reduce or prevent catheter-related blood stream infection (CRBSI), we have to understand the growth properties of the microorganisms that cause this condition. We have previously investigated the growth of the microorganisms that are known as the major causes of CRBSI (Staphylococcus aureus, Serratia marcescen, Bacillus cereus and Candida albicans) in total parenteral nutrition (TPN) solutions without lipid. 10 Therefore, we investigated the growth of the same microorganisms in TPN solutions containing lipid in the present study. In a commercial TPN solution containing lipid (ML), both standard strains and clinical isolates of all microorganisms (S. aureus, S. marcescens, B. cereus and C. albicans) increased rapidly at the original pH of 6.0, even without multivitamins. Although only C. albicans increased equally at any pH value, the growth of S. aureus, S. marcescens and B. cereus was suppressed as the pH value was reduced, with growth halted at pH4.9 (Experiment 1). However, these 3 bacterial species did not increase in another TPN solution con- taining lipid (L+AT1V) even at pH5.5 and pH5.9 (Experiment 3), which is the same result as obtained in the solution without lipid (AT1V or S+AT1V); this finding conflicts with the results of Experiment 1. The conflicting results from these 2 TPN solutions may be attributable to the difference in the bisulfite concen- trations (ML contains NaHSO 3 at a very low concen- tration [20 ppm], but L+AT1V contains NaHSO 3 at a relatively high concentration [400 ppm]) because the bactericidal effect of bisulfite is enhanced in acidic conditions. 21 Therefore, the additional experiment (Experiment 4) was performed to investigate the effect of bisulfite concentration in the TPN solution con- taining both lipid and multivitamins (MLV). As a re- sult, the growth of the 3 bacterial species was sup- pressed or halted at the same pH (5.4 or 5.7) as the concentration of NaHSO 3 increased (20 ppm, 100 ppm, and 200 ppm). These results suggest that the concentration of bisulfite in TPN solutions is an im- portant factor for suppressing bacterial growth, espe- cially between pH5.0 and pH6.0: the bacterial species cannot increase at pH5.9 with 400 ppm of NaHSO 3 , at pH5.7 or pH5.4 with 200 ppm of NaHSO 3 , and at pH5.0 with 20 ppm of NaHSO 3 . Other findings in the present study are as fol- lows: 1) even if lipid is contained, the acidity of TPN solution is the critical factor suppressing the bacterial growth; 2) the addition of lipid enhances mildly the growth of the bacterial species in TPN solutions but does not affect the growth substantially; 3) the addi- tion of multivitamins further enhances the growth of S. aureus but does not affect the growth of S. marces- cens, B. cereus, and C. albicans in TPN solutions con- taining lipid; 4) C. albicans can grow regardless of acidity, bisulfite, and lipid. Because C. albicans could grow at pH5.5 with 400 ppm of NaHSO 3 (AT1V) in our previous study, 10 the effect of bisulfite concentration on the growth of C. albicans was not investigated in the present study. However, it has been reported that C. albicans could not increase in a TPN solution at pH4.4 with 500 ppm of NaHSO 3 , whereas C. albicans increased in the same TPN solution at pH4.4 with 40 ppm of NaHSO 3 or at pH5.0 with 500 ppm of NaHSO 3 . 23 Practically, Candida species can grow rapidly in almost all TPN solutions. The pH values of most of the recent TPN solu- tions are within 5.0 and 6.0, similar to the old TPN solutions. On the other hand, the old TPN solutions contain bisulfite at relatively high concentrations, but the recent TPN solutions contain very low concentra- tions of bisulfite or are bisulfite-free. To investigate bacterial growth in the recent TPN solutions, referring to results from the studies that used the old TPN so- lutions that contained high concentration of bisulfite is not appropriate, even at the same pH range. In the recent TPN solutions containing lipid, some bacterial species may proliferate unless the pH value is 5.0 or less. Although the TPN solutions containing lipid can be theoretically improved to be bacteriostatic by re- ducing the pH value and/or increasing the bisulfite concentration, more studies seem needed to improve the solution because lipid emulsions become unstable as the pH value reduces or as the concentration of bisulfite increases. In conclusion, Candida species can grow rapidly in almost all TPN solutions regardless of the acidity and the presence of lipid; also, some bacterial species may grow in TPN solutions containing lipid unless the pH value is 5.0 or less. Therefore, each TPN solu- tion should be investigated to determine whether or not the bacterial species can proliferate. ACKNOWLEDGEMENTS We are very grateful to Dr. Yoshifumi Inoue, Kawasaki Hospital, Kobe, Japan, for his helpful sug- gestions. Conflict of Interest We declare that there are no conflicts of interest for all of us. References 1. Banton J. Techniques to prevent central venous catheter infec- tion: products, research, and recommendations. Nutr Clin Pract. 2006;21:56-61. Int. J. Med. Sci. 2010, 7 http://www.medsci.org 109 2. Mermel LA, Farr BM, Sherertz RJ, et al. Guidelines for the management of intravascular catheter-related infection. Clin Infect Dis. 2001;32:1249-1272. 3. Llop J, Badia MB, Comas D, Tubau M, Jodar R. Colonization and bacteremia risk factors in parenteral nutrition catheteriza- tion. Clin Nutr. 2001;20:527-534. 4. Allwood MC. Microbiological risks in parenteral nutrition compounding. Nutrition. 1997;13:60-61 5. Didier ME, Fischer S, Maki DG. 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Ishida K, Nakao S, Sata T, et al. Fungistatic action of the total parenteral nutrition (TPN) fluids against clinically isolated Candida albicans. Jpn J Pharm Health Care Sci. 2002;28:259-262. . via intravenous line were recently reported in Japan. 20 In the present study, we investigated the growth in TPN solutions containing lipid of the same microorganisms studied previously: 10 . albicans) in total parenteral nutrition (TPN) solutions without lipid. 10 Therefore, we investigated the growth of the same microorganisms in TPN solutions containing lipid in the present study Candida albicans) in TPN solutions containing lipid. Methods: The pH value of a TPN solution con- taining lipid (pH 6.0, containing 20 ppm of NaHSO 3 ) was adjusted by the addition of HCl to 5.7,