Lysosome Lipid Storage Disorder in NCTR-BALB/c Mice
I. Description of the Disease and Genetics
MANFORD D. MORRIS, PhD, From the Departments ofPediatrics and Biochemistry, University of CHIDAMBARAM BHUVANESWARAN, PhD, Arkansas for Medical Sciences, Little Rock, Arkansas, and the HELEN SHIO, MA, and STANLEY FOWLER, PhD Laboratory of Biochemical Cytology, The Rockefeller University, New York, New York
We describe a strain of BALB/c mice, designated creased, while a-lipoprotein content is decreased. Se- NCTR-BALB/c, carrying a new genetic disorder char- rum total cholesterol remains normal. The serum activ- acterized by excessive tissue deposition of cholesterol ities of asparate aminotransferase, creatine phospho- and phospholipid. The mice exhibit progressive inco- kinase, and N-acetyl-p-glucosaminidase are elevated. ordination, grow less rapidly, and die 80-120 days after Free cholesterol levels are increased 8-10-fold in liver, birth. In comparison with control animals of the same spleen, and thymus, and about 2-fold in other tissues; age, organ weights in the affected animals are lower in but esterified cholesterol levels are normal. The phos- absolute value but higher relative to body weight, ex- pholipid content of several tissues is increased 50-100%, cept for the thymus, which is atrophied, and for the largely as a result of an increase in sphingomyelin con- lung and testes, whose absolute weights are not changed. tent. Significant increases in phosphatidylcholine occur Vacuolated cells are found in many tissues, and large also in spleen and lung. The disorder is inherited, af- foam cells are present in reticuloendothelial system fecting both sexes equaliy, and appears to be transmitted (RES)-rich organs. Compared with those of BALB/c as an autosomal recessive mutation. (AmJ Pathol 1982, controls, serum lipoproteins migrate more slowly on 108:140-149) electrophoresis; the amount of P-lipoproteins is in-
WE HAVE DESCRIBED in preliminary articles"'2 a genetic defect, however, has not been established, strain of BALB/c mice that are affected with an in- and several unusual features of the mouse disorder herited disorder involving progressive neurologic hint at an etiology different from that of typical dysfunction, wasting, and premature death. Tissues Niemann-Pick disease. of the affected mice contained excessive amounts of unesterified cholesterol, and foam cells were seen in liver, spleen, thymus, lung, and gut. A colony of * The strain was derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jeffer- these mice, which we refer to as the NCTR strain,* son, Arkansas. has now been produced, permitting a detailed study Supported by research grants AM-21376 and HL-18157 of the disease. In the papers presented here, we show from the National Institutes of Health and PCM-8008713 that these mice are affected by a generalized lysosome from the National Science Foundation. Dr. Fowler con- lipid storage disorder, and that unesterfied choles- ducted these studies during the tenure of an Otto G. Storm Established Investigatorship from the American Heart terol and sphingomyelin are principal accumulation Association. products. Activities of several lysosomal acid hydro- Accepted for publication March 10, 1982. lases were found increased in tissues of these mice, Dr. Fowler's present address is Department of Pathology, but acid sphingomyelinase activity was decreased. School of Medicine, University of South Carolina, Colum- These findings resemble many of those associated bia, SC 29208. Address reprint requests to Dr. Manford D. Morris, with Niemann-Pick disease in man,3 and they suggest Department of Pediatrics, University of Arkansas for that the NCTR-BALB/c mice could be an experi- Medical Sciences, 4301 W. Markham, Little Rock, Arkan- mental animal counterpart of the disease. The precise sas 72205. 0002-9440/82/0810-0140$01.30 © American Association of Pathologists 140 Vol. 108 * No. 2 LIPID STORAGE DISORDER IN NCTR-BALB/c MICE 141
The evidence supporting our conclusions is re- Animal Sacrifice and Autopsy ported in the accompanying articles,4'5 which The animals were lightly anesthetized with me- both studies on tissues of describe ultrastructural thoxyflurane (Metofane, Pitman-Moore Incorporated, affected NCTR-BALB/c mice and biochemical Washington Crossing, NJ). Blood was obtained from of storage inclusions isolated analyses purified by the heart after opening of the chest. After clotting, subcellular fractionation of livers from these mice. In serum was separated at 40 C, and 5,5'-dithiobis-(2- the present article we describe the main symptoms of nitrobenzoic acid) was added to a portion to inhibit the disorder, including clinical, gross, histopatho- logic, and biochemical findings. Evidence that the serum lecithin cholesterol acyltransferase.7 Organs and tissues were immediately excised, weighed, and disorder is transmitted as an autosomal recessive analyzed while fresh. genetic trait is also presented. While this work was in progress, Pentchev et al6 reported biochemical and enzymic studies on the same strain of mice, and a Histology comparison of their findings with ours is presented in the third article of this series.5 Specimens of tissues were fixed in neutral-buffered formalin and were processed by standard histologic procedures, or were fixed in 2.5 %o glutaraldehyde and Materials and Methods embedded in Epon 812. Thick Epon sections (1 ,) were cut and stained with 0.5% methylene blue, Colony Source 0.5% azure II (Allied Chemical, Morristown, NJ) in All mice described in this report were derived from 0.5%o sodium borate. Photographs were taken with a the strain BALB/cStCrlfC3HfNctr currently in use Zeiss universal photomicroscope on Kodak Pana- at the National Center for Toxicological Research tomic-X film. (NCTR), Jefferson, Arkansas. The propositus mouse (female), discovered in March 1975, was one of Analytic Procedures about 24,000 mice that were used in the 2-ami- noacetylfluorene effective dose 01 experiment con- Enzymes ducted at the NCTR. It was found moribund and ex- Serum aspartate aminotransferase8 and creatine hibited features suggesting a storage disorder (see phosphokinase9 were determined with a Rotochem II Results). The parents of the propositus mouse were a analyzer (Aminco Instruments, Silver Spring, Md). sibling breeding pair; another sibling breeding pair N-acetyl-p-glucosaminidase activity in serum was from the same litter also produced affected progeny. assayed by the method described in the accompany- The NCTR-BALB/c mouse colony described here ing article.5 was developed from these two breeding pairs, pri- marily by brother-sister matings, although early on, Lipids father-daughter matings were also carried out.t The Tissue Extraction: The weighed tissue was homog- control mice were derived from a separate colony ob- enized in a Polytron homogenizer (Brinkman In- tained from the BALB/c breeding stock at the struments, Westbury, NY), and was extracted three NCTR. times with a total of at least 50 volumes of chloro- form-methanol (2: 1). The pooled crude lipid ex- Colony Maintenance tracts were washed and purified by the method of Folch et al. Weanling mice were separated from their mothers Cholesterol: Tissue unesterified and esterified at 21-25 days of age, weighed, identified by toe clip, cholesterol and total serum cholesterol were de- and caged according to sex. Usually, 2-5 animals termined by the method of Rudel and Morris."' For were housed in a single plastic cage. They were allowed tissue total cholesterol, saponification was carried food (Purina Mouse Chow 5015Z) and water ad libi- out in 307o alcoholic KOH at 60 C for at least 30 tum. Affected NCTR-BALB/c mice were detected by minutes. Cholesterol was then extracted and quan- clinical signs and were retained with their littermates. titated by the procedure cited. For measurement of growth, the mice were weighed Phospholipid: Phospholipid phosphorus was de- daily to the nearest 0.1 g on an electronic balance. termined by the method of Fiske and Subbarow.'2 The phospholipid classes were separated by one- t Investigators wishing to acquire these mice should con- dimensional thin-layer chromatography on glass tact Dr. Manford D. Morris. plates coated with silica gel 60 F 254 (Merck and 142 MORRUS ET AL AJP * August 1982
Table 1 -Clinical Signs in Affected NCTR-BALB/c Mice* phosphatidylcholine, and a blank area were scraped Tremor of hind legs when walking and analyzed in every run. Jerky interrupted movements (staggering gait) Front feet spread apart when standing still Serum Electrophoresis Tendency to hold head higher than littermates Outward extension of forelimbs and rhythmic tremor Serum protein and lipoprotein electrophoresis in when held by the tail agarose was performed by a modification of the 13 Listed in order of usual appearance during growth and develop- method of Noble. Serum proteins were stained with ment of mice. 1% aqueous light green SF (Harleco, Philadelphia, Pa), and lipoproteins were stained at 400 C with 0.05% oil red 0 (Sigma Chemical Company) in 700o Company, Cincinnati, Ohio). The solvents used in ethanol. The serum samples were analyzed within 2 succession to separate the classes of phospholipids hours of bleeding. were 1) chloroform/methanol/water (16: 7: 1) and 2) chloroform/methanol/glacial acetic acid/water Results (11 :7.5:2:1).13 A standard mixture consisting of sphingomyelin, phosphatidylcholine, phosphatidyl- Signs and Symptoms of the Disorder inositol, phosphatidylethanolamine, and phospha- Table 1 lists the clinical symptoms exhibited by the tidylserine (Avanti Biochemicals, Birmingham, Ala) affected NCTR-BALB/c mice. The symptoms ap- was included in each run. After detection by iodine peared at variable periods during growth and, once followed by sublimation, the areas corresponding to present, developed progressively in intensity. Tremor the phospholipids were scraped and quantified."4 In of the back legs when walking was often the first symp- addition, the origin, an area corresponding to lyso- tom to appear, usually about 30-35 days after birth.
Figure 1 -Example of neurologic dysfunction in affected NCTR-BALB/c mice at 50-70 days of age. When suspended by the tail, the control BALB/c mouse (left) reflexly stretches forelimbs backwards in an attempt to grasp a surface. In contrast, affected NCTR-BALB/c mouse (right) maintains forelimbs forward and undergoes rhythmic tremors. Also note the arched back, typical of affected mice. These mice are usually smaller than control BALB/c mice of the same age. Vol. 108 * No. 2 LIPID STORAGE DISORDER IN NCTR-BALB/c MICE 143 Males
1% E! L-itl ittPr R
20 -
15 -
Un E 0
Figure 2-Growth patterns of littermate affected (0) 10 -L and unaffected (0) NCTR-BALB/c mice. Four typical examples are shown. The mice were progeny of _-C Females matings that had previously produced affected mice. 0'1 a) Clinical status was confirmed by biochemical criteria 3: at sacrifice. m0
Age in days
Most obvious were the jerky movements and stagger- ing the terminal period; food must be placed directly ing gait that began to occur 4-6 weeks after weaning. in the cages because of the uncoordinated feeding of Another sign was the atypical outward extension the animals. All affected NCTR-BALB/c mice pre- reflex of forelimbs shown by the animals when held maturely die between 80 and 120 days of age. by the tail (Figure 1). These abnormal movements, reflexes, and postures indicate progressive and severe Pathology neurologic dysfunction. The combined appearance of clinical signs is diagnostic for the disorder. At death, the body weights of the affected animals Typical growth patterns of affected and unaffected were little more than half those of unaffected con- sex-matched littermates are shown in Figure 2. trols. Depletion of subcutaneous and mesenteric fat- Affected NCTR-BALB/c mice usually weighed less at ty tissue was very marked, whereas many organs weaning than did their unaffected littermates, and underwent a smaller weight loss or, in the case of this feature has been a reliable predictor of clinical lungs and testes, no significant weight loss at all. signs in the animals. The difference in weaning weight Consequently, when organ weights are expressed as can be as much as 1-2 g (litter B) or less than 0.5 g the percentage of body weight (Table 2), most organs (litter D). For approximately 2 weeks following from the affected animals appear significantly en- weaning, the body weights for affected and un- larged. Exceptions were the heart and diaphragm, affected NCTR-BALB/c mice increase in parallel. which remained unchanged in proportional weight; The affected mice then begin to gain weight less the spleens of females, which were 82% of control rapidly. In the final weeks they lose weight, often values; and the thymus, which was atrophied in both precipitously. The clinical signs are exacerbated dur- sexes. Superficial and visceral lymph nodes were easi- 144 MORRIS ET AL AJP * August 1982
r-0 0 ly visible, and they were about twice their normal C,) 6C5 c; o a) size. Generally, the extent of relative organ C,) +I +I o enlarge- F-a) ._v ment was similar for both sexes. Except for the liver, t00a) which appeared a lighter brown color than normal,
0 the color and texture of the organs from affected E 0 NCTR-BALB/c mice were unremarkable. n 0 a co a) +1 +I Co +1 +l t Alterations in the histologic features of tissues a) 6 from affected NCTR-BALB/c mice were dramatic, (NIo--C) Da ~CDa)'- 0 S'-^ with fatty infiltration in many tissues accompanied E by transformation of reticuloendothelial cells into _4ClC4 V C C) CDC\ m (D foam cells. Examples of tissue sections are illustrated a CD C> C n cm in Figure 3. There were no discernible differences be- a) tween males and females in the distribution and ex- 0 04J'- Nt( 00 00V tent of the lesions. C,) C'¢,- In liver, many large foam cells occurred singly and cn 2E - in clusters; they were present in all lobes (Figure 3A E +1 +l 0 C>+I Cs+I and B), disrupting the cellular architecture of the CJC\- CO tissue. The sinusoids appeared obstructed by red C\- a), ^ blood cells and by the large foam cells. Hepatocytes in control BALB/c liver specimens possessed osmio- C E 0 0 0c c philic whereas in of livers co r-00 00CV o lipid droplets, hepatocytes ._a 8 of affected NCTR-BALB/c mice these droplets were 0' N-co C(0 (5 C-) 0tC) +1 absent, and instead the cytoplasm was finely vac- co0 co £ E0) oo too aE uolated. The spleen was extensively infiltrated with 0 m ,<^ ° large foam cells, most in the red o0C-o Ool 04o K prominently pulp 0) , 8 (Figure 3C and D). Lymphocytes in the white pulp N C +1+1 +I+1 o +1 +1i°+I C 0 _ ta) =talso possessed a vacuolated cytoplasm, but not to the E degree seen in the foam cells. In lung, foam cells were 0 Ct t z ct seen both in the alveolar wall and free in the alveolar c) a co > space (Figure 3E and F). The alveolar space was o6. 66.-. a) 0 0 m 0 V 'D often filled with in contrast to the BALB/c +I +10 +1 +1 ° debris, a O> O O 4 0 E control. Both the cortex and medulla of lymph nodes I O~0 10 CO OC -6 a4C) 04C < -X 0were affected by the fatty infiltration, with extensive 4- cnour C)J 0) cellular vacuolization (Figure 3G and H). The cer- z =cNI vical, axillary, prefemoral, and mesenteric nodes a)C ao'-CD -=, O tO0L
r- U') C0 E was m CVl 4, thymus replaced by foam cells. In kidney, the CD O C) ( -^ CL epithelial cells of proximal tubules were more vacuo-
CD lated than normal. The nerve cell bodies of the ven- - "II Ci C\! tral horn of the 0 a) spinal cord and of the brain stem ._ +I +I +I +I 0 -j (DX,4) " possessed a finely vacuolated cytoplasm. In the cere- 0 0 C a)) bellar cortex, a depletion of cells in the Purkinje layer xD)2 z - - - CM _L a ,was evident both at the tip of the gyri as well as deep 04 0. 2 CD in the sulci; the molecular layer contained foci of _- C) _. Q 20 cCO (1 cCO)@ao CO CD =
Table 3-Cholesterol and Selected Enzyme Levels in Serum of Control BALB/c and Affected NCTR-BALB/c Mice* Constituent Total Aspartate Creatine N-acetyl-p- Sex Group cholesterolt aminotransferaset phosphokinaset glucosaminidaset Male Control 176 + 20 (6) 45 ± 10 (4) 131 ± 30 (4) 8.5 ± 2 (4) Affected 151 ± 20 (6) >500 (4) 311 ± 150 (4) 29.9 ± 4 (4) A/C§ 0.86 >11 2.4 3.3 Female Control 115 ± 10 (6) 50 ± 10 (4) 250 ± 150 (4) 14.5 ± 3 (4) Affected 131 ± 20 (6) 366 ± 80 (4) 334 ± 80 (4) 35.0 ± 10 (4) A/C 1.1 7.3 1.3 2.8 Data are given as mean ± SD followed by parentheses giving number of animals studied. t Concentration expressed as milligrams per deciliter. t Enzyme activity expressed as milliunits per milliliter serum. § Ratio of means of affected and control serum concentrations. Vol. 108 * No. 2 LIPID STORAGE DISORDER IN NCTR-BALB/c MICE 147
Table 4-Cholesterol and Phospholipid Content of Selected Organs of Control BALB/c and Affected NCTR-BALB/c Mice* Cholesterol/ Organ Free cholesterol Esterified cholesterol Total phospholipid phospholipidt Liver Control (C) 2.4 ± 1 0.67 ± 0.2 37.9 ± 3 0.127 ± 0.01 Affected (A) 27.2 ± 7 0.59 ± 0.4 52.7 ± 8 1.024 ± 0.12 A/C* 9.0 0.88 1.4 Spleen Control (C) 3.4 ± 1 0.16 ± 0.1 18.3 ± 1 0.370 ± 0.03 Affected (A) 25.4 ± 1 0.12 ± 0.2 41.7 ± 5 1.302 ± 0.07 A/C 7.7 0.75 2.3 Kidney Control (C) 4.3 ± 1 0.11 ± 0.03 37.9 ± 1 0.229 ± 0.03 Affected (A) 11.9 ± 2 0.11 ± 0.1 42.4 ± 2 0.565 ± 0.05 A/C 2.7 1.0 1.1 Lung Control (C) 5.3 ± 1 0.34 ± 0.1 28.7 ± 2 0.370 ± 0.03 Affected (A) 15.5 ± 2 0.24 ± 0.1 58.8 ± 5 0.529 ± 0.04 A/C 2.8 0.71 2.0 Cerebellum Control (C) (14.3 ± 1)§ - 50.6 ± 4 0.567 ± 0.03 Affected (A) (15.3 ± 1)§ - 48.5 ± 3 0.634 ± 0.02 A/C 1.1 1.0 Data are given as milligram per gram tissue wet weight and expressed as mean ± SD of 4 animals in each group. Values for males (2) and females (2) in each group were similar and were pooled. t Molar ratio of free cholesterol to total phospholipid; assumed molecular weights: cholesterol, 386; phospholipid, 775. t Ratio of mean content of affected and control tissues. § Total cholesterol.
cholesterol levels were increased in the same propor- been made. We do not have a test for detecting tion (Tables 4 and 5). heterozygotes except through matings.
Inheritance of the Disorder Discussion The circumstances in which the propositus was Although a variety of mutants of mice have been found-namely, the progeny of a sibling pair with reported with neuropathies related to that observed common ancestors several generations back, and the in the affected NCTR-BALB/c mice, few have an as- production of male and female affected mice by sib- sociated visceral disease as described here."5 A mu- ling pairs of the same litter as the propositus parents tant strain of CBA mice (fm/fm) exhibiting foam cell -suggested that the disorder might be due to an reticulosis was described by Lyon et al in 1965.16 The autosomal recessive gene(s). Examination of the primary tissues affected were thymus and Peyer's transmission of the trait in our NCTR-BALB/c patches, which were greatly enlarged. In addition, mi- mouse colony over the past 2 years, summarized in nor foam cell infiltrations were seen in lymph nodes Table 6, confirms this notion. Of 1591 progeny derived and in other tissues. Fredrickson et all' found that the from 61 sibling mating pairs, 16.2% exhibited the levels of cholesterol in the thymus of the fm/fm mice clinical traits described here. Affected males slightly were elevated 3-fold and that the sphingomyelin con- outnumbered affected females. The portion of tent of the same tissue was increased 7-fold. The lipid affected NCTR-BALB/c mice in first litters was content of the liver and spleen of these mice was 21 %. In subsequent litters, however, the percentage reported normal, although in another report Adachi of affected mice consistently dropped to 16% or less. et al'8 found some increases in phospholipid and The average litter size at weaning remained constant, cholesterol. Sphingomyelinase activity in thymus, and we do not know what accounted for the decrease liver, and spleen was normal. Fredrickson et all' in the number of affected animals produced in later concluded that the foam cell reticulosis exhibited by litters. We have not yet succeeded in breeding any of the fm/fm mice was chemically analogous to those the affected NCTR-BALB/c mice, and thus a forms of human sphingolipidoses not accompanied rigorous test for an autosomal recessive gene has not by a decrease in tissue sphingomyelinase. Differences 148 MORRIS ET AL AJP * August 1982
Table 5-Phospholipid Composition of Selected Organs of Control BALB/c and Affected NCTR-BALB/c Mice* Phosphatidyl- Phosphatidyl- choline and serine and Total lysophosphatidyl- Phosphatidyl- phosphatidyl- Organ phospholipidt Sphingomyelin cholinet ethanolamine inositol Liver Control (C) 37.9 + 3 1.6 ± 0.2 19.8 + 2 12.1 ± 1 4.5 ± 0.5 (4 ± 1)§ (52 + 1) (32 ± 1) (12 ± 1) Affected (A) 52.7 + 8 13.3 ± 4 20.8 ± 2 14.3 + 3 4.4 ± 0.5 (25 ± 3) (40 ± 3) (27 + 2) (8 ± 1) A/C 1.4 8.3 1.1 1.2 0.97 Spleen Control (C) 18.3 ± 1 1.5 ± 0.2 8.5 ± 1 5.2 ± 0.2 2.6 ± 0.3 (9 ± 1) (47 ± 3) (31 + 2) (15 ± 1) Affected (A) 41.7 ± 5 13.5 ± 2 14.4 ± 0.4 5.8 ± 1 3.2 ± 0.2 (33 ± 2) (36 ± 3) (22 + 5) (10 ± 2) A/C 2.3 8.8 1.7 1.1 1.2 Kidney Control (C) 37.9 ± 1 4.1 ± 1 15.8 ± 1 13.5 ± 0.4 4.7 ± 0.4 (11 ± 1) (42 ± 2) (36 + 1) (13 ± 1) Affected (A) 42.4 ± 2 6.7 ± 1 17.1 ± 1 13.8 ± 0.2 4.7 ± 0.2 (16 ± 2) (40 + 2) (33 + 2) (11 ± 1) A/C 1.1 1.6 1.1 1.0 1.0 Lung Control (C) 28.7 ± 2 2.9 ± 0.2 14.3 ± 1 7.8 + 1 3.9 ± 0.3 (10 ± 0) (50 ± 1) (28 ± 0) (12 ± 0) Affected (A) 58.8 ± 5 5.3 ± 1 39.5 ± 4 10.0 + 1 4.1 ± 0.2 (9 ± 2) (67 ± 2) (18 ± 0.5) (7 ± 1) A/C 2.0 1.8 2.8 1.3 1.2
* Data are derived from same animals presented in Table 4 and are given as milligrams per gram wet weight. They are expressed as mean + SD. t Same data as presented in Table 4. t Includes small amount of unidentified material remaining at origin of TLC plate. § Percentage of total phospholipid. Phosphate recoveries from TLC plates ranged from 76% to 98% (mean, 91 %). II Ratio of mean content of affected and control tissues.
in age of onset, clinical symptoms, longevity, and ex- al drugs,24"25 but increases in cholesterol comparable tent and scope of tissue involvement clearly differen- to those found in the affected NCTR-BALB/c mice tiate the fm/fm mice from the NCTR-BALB/c mice are not found. described here. The most striking features of the disorder affecting Sphingomyelin and cholesterol tissue deposition the NCTR-BALB/c mice are the progressive neuro- have also been reported in cats,19"2 a poodle,22 and pathy, vacuolation of parenchymal cells, and appear- shell parakeets.23 A severe inherited neurovisceral ance of foam cells and concomitant elevation of disorder was described for each. In most cases, how- cholesterol and sphingomyelin in several tissues. ever, only formalin-fixed tissue has been examined. These are properties common to various sphingolipi- Tissue accumulations of phospholipids, including doses in humans designated under the collective sphingomyelin, occur in rats after treatment by sever- name Niemann-Pick disease.3 To determine whether
Table 6-Inheritance by Litter of Affected Traits in NCTR-BALB/c Mice* Successive litterst 1st 2nd 3rd 4th 5th 6th Average litter sizet 5.8 + 2 (61) 6.6 + 2 (59) 5.7 ± 3 (53) 6.1 + 2 (44) 5.9 + 2 (32) 4.6 + 2 (20) Total weaned mice 354 388 301 268 188 92 Number of affected males 43 34 26 25 9 6 Number of affected females 30 29 21 16 12 7 % affected mice 20.6 16.2 15.6 15.3 11.2 14.1 Affected mice defined by clinical signs given in Table 1; in most animals, affected status verified by determination of cholesterol content of liver (>5-fold increase above control liver). Number of progeny produced by all breeding pairs are pooled for each successive set of litters. t Each heterozygote breeding pair produced 1 to 6 litters. t Data given as mean + SD with number of heterozygote breeding pairs in parentheses. Vol. 108 * No. 2 LIPID STORAGE DISORDER IN NCTR-BALB/c MICE 149 the NCTR-BALB/c mice represent a model of one of for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957, 226:497-509 these human disorders or are the result of some other 11. Rudel LL, Morris MD: Determination of cholesterol process, one needs more detailed morphologic and using o-phthalaldehyde. J Lipid Res 1973, 14:364-366 biochemical studies. Such studies are presented in the 12. Fiske CH, Subbarow Y: The colorimetric determina- tion of phosphorus. J Biol Chem 1925, 66:375-400 accompanying articles.4 5 13. Lee JA, Morris MD: Characterization of the serum low- density lipoproteins of normal and two Rhesus mon- keys with spontaneous hyperbeta-lipoproteinemia. Bio- chem Med 1974, 10:245-257 14. Skipski VP, Peterson RF, Barclay M: Quantitative analysis of phospholipids by thin-layer chromatogra- References phy. Biochem J 1964, 90:374-378 15. Sidman RL, Green MC, Appel SH: Catalog of the 1. Morris MD, Bhuvaneswaran C, Boothe AD: Tissue Neurological Mutants of the Mouse. Cambridge, Har- cholesterol disorder in BALB/c mice (Abstr). 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