Lipid phenotypes, apolipoprotein genotypes and cardiovascular risk in nonagenarians

Atherosclerosis, 83 (1990) 137-146 Elsevier Scientific Publishers Ireland, Ltd.

137

ATHERO 04499

Lipid phenotypes, apolipoprotein genotypes and cardiovascular risk in nonagenarians

Sheldon L. Thieszen I, James E. Hixson 2, Delwyn J. Nagengast 3, Janet E. Wilson 1 and Bruce M. McManus ’

’ Curdmvascular Registry, Department of Pathology and Microhlology, Universi[v of Nebraska Medical Center, Omaha, NE (U.S.A.), -’ Departmenl of Genetics, Southwest Foundation for Biomedical Research, San Antomo, TX (U.S.A.), and ’ Bloomfield Clinic,

Bloomfield, NE (U.S.A.)

(Received 15 August, 1989) (Revised, received 15 March, 1990)

(Accepted 19 March, 1990)

Despite great interest in the role of lipids in overall and disease-free survival, virtually no information is available on the lipids, lipoproteins and apolipoproteins of persons over 90 years of age. Furthermore, the genetic underpinnings of atherosclerosis and the particular genetic factors responsible for protection against coronary artery disease remain speculative. In Bloomfield, Nebraska, we studied 41 nonagenarians (10 males, 31 females), with a mean age of 92.7 years. in whom lipids, lipoproteins, apolipoproteins and restriction fragment length polymorphisms (RFLPs) of genes for apolipoprotein B (apo B), aop AI and apo CIII were assessed. Nearly complete historical, physical and laboratory data were obtained on 39 subjects. The mean diastolic and systolic blood pressures for this group were nonhypertensive, body mass indices (weight/ height2) had a mean of 23.9 and triceps skinfold thickness measurements an overall mean of 14.8 mm. The mean total serum cholesterol was 5.42 mmol/l. HDL-cholesterol levels in females persisted to be higher when compared to males (P < 0.013). The allele frequencies for apo AI (MspI and PstI), apo CIII (St) and apo B ( XbaI) gene RFLPs were typical for larger population studies. In these preliminary studies, we did not identify a distinctive phenotype, genotype, or phenotype-genotype relationship. Diversity of cardiovascular risk was the hallmark of these nonagenarians.

Key words: Nonagenarian; Risk factors; Diet; Lipids; Lipoproteins; Apolipoproteins; Longevity; Re- striction fragment length polymorphism; Body mass index; Triceps skinfold

Introduction Correspondence to: Bruce M. McManus, M.D., Ph.D., Di-

rector, Cardiovascular Registry, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-3135, U.S.A.

The average life expectancy of United States citizens has increased steadily during the course of

0021-9150/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland, Ltd.

138

the present century. In 1985 life expectancy for men and women was 71.5 and 78.8 years, respectively, and it is projected to reach 75.0 years for men and 83.1 years for women by the year 2040 [l]. Greater life expectancy and increasing median age of survival in the United States derive from improved public health measures and the general quality of nutrition, as well as from diagnostic and therapeutic advances.

Reduced risk for coronary atherosclerosis has been emphasized as a factor which may contribute to longevity. Indeed, the ‘longevity syndrome’, a situation wherein family members of a cohort uniformly live to old age, has been related to low serum levels of low density lipoprotein (LDL)- cholesterol and elevated levels of high density lipoprotein (HDL)-cholesterol [2-51. The degree to which this same set of blood lipid characteristics explains the difference in survival of men and women remains unclear [6,7].

Interest in the role of lipids in overall and disease-free survival to old age has not eventuated in substantive data on this segment of the population. The prevalence database from the Lipid Re- search Clinics includes all subjects above 70 years of age in a single group for determination of percentile categories for different lipid parameters [8]. In addition, the data were obtained over fif- teen years ago. Thus, age-related documentation of lipid and lipoprotein levels has typically extended only to the seventh decade, and rarely to the ninth decade [9]. Apolipoproteins have received even less attention in the elderly. Lack of available information on subjects in their ninth and tenth decades of life [4], is perhaps based on the assumption that little insight can be gained from study of longevity at longevity.

As implied, the genetic underpinnings of longevity are yet unknown, and the particular genetic factors responsible for protection against coronary atherosclerosis remain speculative. Sev- eral investigators have applied molecular tools to subjects or patients to define the “risk-related” allelic frequencies of lipoprotein receptor genes or apolipoprotein genes as indicated by restriction fragments (for review see refs. 10, ll), but none have considered whether certain restriction fragment length polymorphisms (RFLPs) typify long survival.

The recent Working Conference on the Recog- nition and Management of Coronary Heart Dis- ease in the Elderly [12] has reemphasized the need to understand the epidemiology and pathophysi- ology of heart disease in elderly persons. Concern about the nutritional status of the elderly [13] juxtaposes the problems of excessive caloric intake and malnutrition in this segment of the population. However, correlative data illustrating the association of dietary and social factors with blood lipid parameters in people over 90 years of age are virtually non-existent.

The intent of the present study was therefore threefold: (1) to characterize the lipid, lipoprotein apolipoprotein phenotypes of very long survivors, (2) to determine if “protective” genotypes for RFLPs in apolipoprotein genes (apo AI, B, and CIII) can be identified that account for their longevity, and (3) to relate the phenotypic profiles to personal and dietary factors.

Methods

Patient population Nearly 10 000 people in the State of Nebraska

are over 90 years of age [14]. Subjects participat- ing in the present study reside in Knox County in northeastern Nebraska and represent a sample from the rural communities of Bloomfield, Creigh- ton, Niobrara, Center and Verdigre. The study group was identified from the clinical practice of Dr. Nagengast.

A short article was printed in local newspapers explaining the general significance of diet and cholesterol, the current trends toward increasing longevity, and the purpose of the study, and included an invitation to participate. Contact with nursing homes was also made seeking the involve- ment of nonagenarians under Dr. Nagengast’s care. A total of 66 potential subjects were person- ally contacted for inclusion in the study, and of these, 41 agreed to participate. The final sample included 31 females and 10 males; 26 subjects lived in a residential home, while 15 were of independent living status.

Data collection Participants in the study provided informed

consent on an IRB-approved form and were subsequently scheduled for collection of physical, di-

139

etary and laboratory data. The nursing home and clinical staff assisted in scheduling and data collection. In the nursing homes, staff nutritionists were instructed to record each participant’s dietary intake for a 24-h period preceding data collection, while those living independently re- corded this information themselves or with the assistance of a family member. Upon consent, each subject’s medical records were extensively reviewed to obtain current and historical medical data to supplement that provided directly by each subject.

Historical data sought from each nonagenarian included level of education, ancestry, marital status, work status and exercise. Self-reported dietary data included rating of personal diets on levels of fat, cholesterol, and salt, and an indica- tion of responsibility for diet and cooking, as well as, information on bowel conditions, use of laxatives, vitamins and minerals, dietary restrictions, food allergies, difficulties with chewing and/or swallowing and presence of dentures. Data obtained also included current medical therapy and a perception of responsibility for health, personal interpretations of blood pressure and cholesterol levels, history of smoking and alcohol consumption, and cardiovascular symptoms.

Physical measurements of systolic and diastolic blood pressure (mm Hg), height (cm), and weight (kg) were taken by standard methods and a mea- surement of triceps skinfold thickness (mm) was obtained by a QuintonTM caliper. Values for body mass index (BMI) were determined using the equation BMI = weight (kg)/height (m*), and provided a complementary estimate of ponderos- ity [15,16]. In addition, subjects were evaluated for the presence of cornea1 arcus, xanthomas and xanthelasmas.

Dietaty analysis Dietary data were obtained in the manner de-

scribed above, and subsequently the Practorcare software program, NutripractorTM, was utilized for dietary evaluation of the dietary recalls. This nutrient analysis program is based on data from the United States Department of Agriculture (USDA) Handbook No. 8, as well as, additional data from the food industry and the USDA. All nutrient data from the dietary recalls were

analyzed and transferred to the UNMC IBM 3090 mainframe computer for statistical analysis. The food frequency questionnaires were manually in- terpreted by a registered dietitian and entered into the mainframe. Each nonagenarian’s dietary recall was assessed for nutrient composition including total calories, carbohydrates, proteins, total fat, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, mg cholesterol/1000 kcal and the polyunsaturated/ saturated fat ratio.

Serum analysis A venous blood sample was obtained following

a lo-12 h fast, and was utilized for analysis of total cholesterol, lipoproteins, and apolipoproteins, and for apoprotein RFLPs from DNA of peripheral blood mononuclear cells. A total of 3 tubes were drawn from each patient: two lo-ml lithium-heparin tubes and one 15ml separator tube. The separator tubes were centrifuged and express-mailed to MetPath Laboratory, Teterboro, NJ. This laboratory was responsible for analysis of serum lipids, lipoproteins, and apolipoproteins and has fulfilled standardization requirements of the CDC Laboratory Standardization Program for serum cholesterol determination. The enzymatic method for determining total cholesterol was carried out on a Prisma autoanalyzer. Lipoprotein analysis included HDL-cholesterol (phosphotung- state precipitation on a KDA monitor), VLDL- cholesterol (triglycerides/5), and calculated LDL- cholesterol, the latter being quantified by the Friedewald equation [17]. Apolipoproteins, AI and B, were quantitated on a Behring analyzer by rate immunonephelometry [18,19].

The two lo-ml heparinized tubes were shipped overnight to the University of Nebraska Medical Center for harvest of peripheral blood mononuclear cells for RFLP analysis, and for immuno- logical studies to be reported elsewhere [20]. The mononuclear cells were isolated from heparinized blood with LymphoprepTM at 800 x g for 15 min. The isolated cells were then stored at -70°C in cryovials with a solution of 65% RPM1 medium containing penicillin-streptomycin. Hepes buffer, 20% fetal calf serum, and 15% dimethylsulfoxide, and were shipped on solid CO, to Dr. Hixson, at the Southwest Foundation for Biomedical Re- search, San Antonio, TX.

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RFLP analysis High molecular weight DNA was prepared from

leukocytes purified on Sepracell-MN gradients (Sepratech Corporation) and lysed with SDS (5%) and proteinase K (100 pg/ml) at 55” C for 16 h [21]. After phenol extraction and ethanol precipitation, DNA was resuspended (1 mg/ml) for further analysis. For Southern blots [22], purified DNAs were digested with restriction enzymes, electrophoresed on agarose gels (OX!%), and transferred to nylon membranes in alkaline buffers as previously described [23]. After prehybridization (4 h in 50% formamide at 42” C), the membranes were hybridized with radiolabeled probes [24], and washed at high stringencies for autoradiography. The probes used in this study were a human apo AI cDNA clone (provided by Dr. Jan Breslow; [25]) and a 5.8 kb SalI-BamHI genomic fragment from the 3’ coding region of the apo B gene (provided by Dr. James Scott; [26]).

Statistical analysis All data were entered on the UNMC IBM

model 3090 mainframe computer using the VM/CMS 5.0 operating system. Descriptive sta- tistics were prepared using the SAS version 5.18, for the overall group and according to subgroups based upon gender, education, level of living inde- pendence, BMI and dietary intakes. A multiple correlation matrix was prepared to assess environ- mental and genetic factors predictive of cholesterol and other lipid and lipoprotein levels. Tests of difference were utilized to compare study subgroups with respect to phenotypic lipid, lipoprotein, and apolipoprotein parameters and to evalu- ate the lipid phenotype-apolipoprotein genotype associations. Statistical significance was set at the 0.05 alpha level.

Results

Of the 41 subjects in the study, one patient had polycythemia rubra vera and another had chronic lymphocytic leukemia. These 2 patients were ex- cluded from the database. Nearly complete historical, physical and laboratory data were obtained on the remaining 39 patients (10 males and 29 females). At the time of the study, all subjects

were on their usual diet and had reported no recent substantial changes in body weight. Satis- factory DNA from peripheral blood mononuclear cells was obtained for RFLP assessment in 23 of the 39 subjects (4 males and 19 females).

The mean subject age was 92.7 years (range 90-98 years), the mean for males and females being 92.3 and 92.9 years, respectively. A majority of the subjects reported German ancestry (i.e., 22 of 32 responses) while an additional 6 subjects were of Anglo-Saxon origin and 2 were of Slavic background. Of 24 responses regarding educational level achieved, 6 subjects reported a 6th grade level, 12 an 8th grade level and 5 an educational level greater than the 8th grade. With re- gards to alcohol consumption, 26 subjects responded that they had never consumed alcoholic beverages and 6 reported that they still drank or that they had stopped over a year ago. All female subjects responding to questions regarding smoking, stated that they had never used tobacco products. Four male subjects were currently smoking lo-19 cigarettes per day with a history of 10 or more years of this activity. The other 6 male subjects responded that they had never smoked. Of the 39 subjects, 29 had at least partial dentures (7 males and 22 females). Nineteen subjects reported the use of laxatives. There was only a single case of food allergies reported and this was to dairy products. Six of the 39 subjects used vitamin or mineral supplements on a regular basis, 32 subjects reported a salt restricted diet and 28 subjects had meals prepared by an institution (10 by themselves or family member). In most cases the subjects considered their diet intake of fat and cholesterol as normal. When questioned about marital status, 32 subjects responded that they were widowed, 4 were married (3 males and 1 female), and 3 were single or divorced.

Information regarding medications and general cardiovascular health status was available in the 26 nursing home residents. Of these subjects 21 carried a diagnosis of atherosclerotic coronary disease, 6 of whom were also suspected to be in congestive heart failure. In addition, 3 subjects had a history of stroke. Digoxin was prescribed in 12 subjects, diuretics in 14, and beta-adrenergic blockers in 3. A majority of the subjects (24 of 39) reported regular exercise.

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TABLE 1

PHYSICAL CHARACTERISTICS OF 39 NONAGENARIANS

Subjects Age Height

(yrs) (m)

Weight

(kg)

BMI Triceps skinfold (mm)

Blood pressure (mm Hg)

Diastolic Systolic

Ml& Mean

(SD) Range

Female Mean

(SD) Range

OV~IZN Mean

(SD) Range

92.3 1 .I2 14.4 25.2 12.1

(k1.1) (+O.l) (+11.3) (*4.5) (f4.2) 91-94 1.60-1.85 60.1-100.0 20.4-35.6 7-20

92.9 1.59 59.5

(*2.3) (kO.1) (i11.0) 90-98 1.27-1.80 39.6-81.8

92.1 1.63 63.3

(k2.1) (iO.l) (* 12.8) 90-98 1.27-1.85 39.6-100.0

23.5 15.7 (*4.5) (k7.8)

16.7-30.6 4-32

23.9 14.8 (* 4.5) (k7.2)

16.7-35.6 4-32

75 133 (i 10.0) (k 14.5)

60-90 120-168

68 122 (k 8.2) (f15.3)

54-84 104-158

70 124

(i-9.1) (k15.7) 54-90 104-168

Physical characteristics Physical characteristics of 39 nonagenarians are

summarized in Table 1. The mean diastolic and systolic blood pressures for the overall group were 70 and 124 mm Hg, respectively. Neither blood pressure parameter was significantly different between males and females, with a diastolic mean of 68 and 75, and a systolic mean of 122 and 133 mm Hg, for females and males, respectively. Of note, the diastolic pressures were all nonhypertensive ranging from a minimum of 54 to a maximum of 90 mm Hg, while the systolic pressures ranged from a minimum of 100 to a maximum of 168 mm Hg. However, only 2 subjects had a systolic blood pressure greater than 150 mm Hg.

The mean BMI was 25.2 and 23.4 for males and females, respectively, these indices ranging from as low as 16.7 to as high as 37.2. The frequency distribution curves of BMI values for both males and females are near Gaussian with rightward extended tails. A BMI value of 22-27 is considered to be the range for normal, with the upper limit of 27 being borderline obesity [27]; there are 7 subjects in this study above this level. The mean triceps skinfold measures were 12.1 and 15.7 mm for males and females, respectively. Again, a wide range in values was noted, from 4.0 to 32.0 mm. The mean values for males and females were comparable to the 25th percentile of norms in white subjects aged 80-89 years reported by Falciglia et

al. [28] and similar to means for males and females over 90 years of age reported by Campbell and Borrie [29] as 14.7 and 14.8, respectively. In this sample of nonagenarians, the subjects were generally of short stature, at 1.72 m and 1.59 m for males and females, respectively.

When subjects were subdivided into those above versus those below the mean BMI, it was observed that subjects with values below the mean had significantly lower skinfold thickness (P = 0.002) and LDL-cholesterol levels (P = 0.04). Trends toward significant differences in these BMI groups were also noted for levels of total cholesterol (P = 0.09) HDL-cholesterol (P = 0.11) and the ratio of LDL-cholesterol to apolipoprotein B (P = 0.06).

Dietary intake The mean caloric intake for 38 males and

females was 1779 kcal/day. The intake varied dramatically from 353 to 3373 kcal/day. Males and females were not statistically different in their local caloric intake with a mean of 1886 and 1746 kcal/day, respectively. Greater variability was noted within the female group than in the males. The diet of these 38 nonagenarians was relatively high in carbohydrates with the percent of dietary calories due to carbohydrates at 53%, while protein and fat were 16.6% and 31.9%, respectively. The consumption of carbohydrates was signifi-

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# SUBJECTS

4.0 4.8 56 6.4 7.2 8.0 0.6

- TC 0’nmol/L)

Fig. 1. Frequency distribution of total serum cholesterol (TC) in 39 nonagenarians. The number of subjects are plotted versus TC in mmol/l. The mean TC for these subjects was 5.42 mmol/l, ranging from 3.88 to 8.12 mmol/I. The two vertical lines (a) and (b) represent NCEP risk cutpoints of 5.17 and

6.21 mmol/l, respectively.

cantly higher in females than in males at 54% and 47% of total calories, respectively (P = 0.006). In addition, the females consumed fewer calories as fats, 30.5% in comparison to 36.4% of total kcal (P = 0.007). Protein intake was similar in gender groups at 17.1% and 16.5% of total kcal in males and females, respectively. The polyunsaturated to saturated fat ratio of calories in the diet was rather low for both males and females at 0.40 and 0.35, respectively. These values, however, were not statistically different. The percent of dietary calories due to saturated fat, monounsaturated fat and polyunsaturated fat were also not different between males and females. Similarly, the mean daily cholesterol consumption at 207 and 211 mg

for males and females, respectively, was not statistically different.

Lipid phenotype As noted in Table 2, the mean blood cholesterol

level in 39 subjects was 5.42 mmol/l. Males and females were not different in their mean levels at 5.35 and 5.45 mmol/l, respectively. The values ranged from 3.88 to 8.12 mmol/l. Four male and 8 female subjects exceeded the level designated by NCEP as borderline high, while 10 subjects would be considered at “high risk” (Fig. 1). Similar diversity of lipid parameters was seen for triglycerides, LDL-cholesterol, apolipoproteins AI and B, and various ratios of the lipids, lipoproteins and apolipoproteins (Table 2). HDL- cholesterol was significantly lower in male subjects, with a level of 1.02 mmol/l, than in females, with a level of 1.20 (Fig. 2). Apo AI levels ap- proached a significant difference (P = 0.16) when comparing genders with males having a value of 114.4 and females 124.8 mmol/l. Mean values for the lipid, lipoprotein and apolipoprotein ratios are given in Table 2. These ratios did not significantly differentiate males from females. Educational status or ancestry did not appear to bear on the physical, dietary or blood lipid and lipoprotein parameters.

Injluence of living status Consideration of independent versus residential

home living status revealed significant differences

TABLE 2

LIPID, LIPOPROTEIN AND APOLIPOPROTEIN PHENOTYPES (mmol/l) OF 39 NONAGENARIANS

Subjects

Male Mean

(SD) Range

Female Mean

(SD) Range

Overall Mean

(SD) Range

TC HDL-C

5.35 1.02

(+l.o) (kO.1) 4.03-7.40 0.88-1.19

5.45 1.20

(+l.l) (rtO.3) 3.88-8.12 0.80-1.89

5.42 1.15

(+l.l) (*0.3) 3.88-8.12 0.80-1.89

LDL-C

3.22 (+1.3)

2.26-5.54

3.25

(kl.0) 1.67-5.45

3.25

(*l.o) 1.67-5.54

Trigs

2.41 ( + 2.4)

0.81-9.09

2.18

(*lo) 0.90-4.09

2.24

(+1.5) 0.81-9.09

Apo AI

114.4 (+ 15.8)

94.5-153.0

124.8 (+21.1)

92.2-185.4

122.1 (+ 20.2)

92.2-185.4

ApoB

98.8 (+ 31.0)

53.3-168.0

92.7 ( + 28.2)

42.8-154.5

94.3 (+ 28.6)

42.8-168.0

8 SUBJECTS 35

. .

‘. ‘~

158 178

HDL-C (mmol/L)

-MALES -- FEMALES

Fig. 2. Frequency distribution of HDL-cholesterol (HDL-C) for 10 male and 29 female nonagenarians. Percent of each gender is plotted versus HDL-C in mmol/l. The conformation, mean and range of the frequency distribution for females is

distinctively different than that for males.

between these subject groups on several parameters. Mean systolic blood pressure was significantly lower (P = 0.001) in residential home subjects than in those living independently at 118 and 138 mm Hg, respectively. In addition, mean diastolic blood pressure was lower in the institutionalized subjects at 66 versus 78 mm Hg diastolic blood pressure (P = 0.0001). The independent living subjects also consumed significantly fewer calories, 1070 kcal/day, than subjects in residential homes, 2068 kcal/day (P = 0.001). The percent of calories as protein was significantly greater in the independent living subjects than the residential home subjects at 18.4% and 15.9%, respectively (P = 0.02). Other physical and dietary parameters were not different between the 2 groups. Where lipid parameters were concerned, the only difference between the institutionalized and independent living subjects was in the apo AI levels, which were higher in the institutionalized

TABLE 3

143

subjects at 166.7 mmol/l compared to the free living subjects at 143.3 mmol/l (P = 0.005).

Genotypic analysis Twenty-three nonagenarians were typed for

RFLP genotypes at loci encoding proteins of cholesterol metabolism to determine if “protective” genotypes could be identified that account for their longevity. Genotypes for particular RFLPs in apolipoprotein genes (apo AI, B, CIII) were selected for which significant effects on lipid levels or heart disease had been detected in previ- ous studies (for review, see refs. 10, 11). The results of RFLP analysis for the apo AI-C111 gene cluster in nonagenarians are shown in Table 3. Using these data, the allele frequencies for each RFLP were calculated to determine if gene frequencies were different in these nonagenarians reflecting effects of ‘protective’ genotype. The frequency of the rare allele (M2) for MspI cleavage in intron 3 of the apo AI gene was 0.05 in these nonagenarians. This M2 frequency is very similar to those from several studies of Caucasian populations that range from 0.03 [30] to 0.11 [31]. The frequency of the rare allele (P2) for PstI cleavage 3’ to the apo AI gene was 0.02, also similar to those for Caucasian populations [32]. The frequency of the rare allele (S2) for SstI cleavage downstream from the apo CIII gene (0.02) did not differ from allele frequencies from Cauca- sian populations [30].

These results indicate that nonagenarians are not distinguished by particular allelic frequencies for RFLPs in the apo AI-C111 gene cluster. RFLP genotypes were also determined for a polymorphic XbaI site located in the large exon at the 3’ end of the apo B gene. The apolipoprotein B gene has

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs) IN THE GENES FOR APOPROTEINS AI. CIII AND B IN 23 NONAGENERIANS

RFLPs were not obtained in all subjects with each restriction enzyme; thus, the total of each gene-restriction site combination does not add up to 23. Number of subject with each polymorphisms is given in parentheses.

Gene restriction site: ApoAI- MspI ApoAI- Psr I

MlMl (19) PlPl (21) M2M2 (1) PlP2 (1)

ApoCIII-SsrI

SlSl (21) SlS2 (1)

ApoB- Xba I

x1x1 (7) x1x2 (9) x2x2 (5)

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TABLE 4

RELATIONSHIP OF LIPID PHENOTYPES (mmol/l) TO XbaI RESTRICTION SITE OF THE APO B GENE 21 NON- AGENARIANS

Evaluated by General Linear Models Procedure

Phenotype Genotype P

x1x1 x1x2 x2x2 value

(n=7) (n=9) (n=5)

Total cholesterol

HDL-C LDL-C Triglycerides Apo AI Apo B TC/HDL-C LDL-C/HDL-C Apo B/ape AI LDL-C/ape B

5.51 5.75 5.10 > 0.52 1.27 1.23 1.13 > 0.68 3.40 3.60 2.57 > 0.25 1.84 2.02 3.06 > 0.49

162.05 148.25 157.10 > 0.62 149.87 171.96 184.22 > 0.55

4.07 4.48 4.44 > 0.85 2.33 2.59 1.75 > 0.47 0.801 0.990 0.995 > 0.50 1.175 1.090 0.857 > 0.10

been extensively studied and the sites of X&I cleavage previously mapped [26]. Calculated allele frequencies for the Xl and X2 alleles (from the genotype determinations shown in Table 3) were 0.55 and 0.45, respectively. Similar to the results for apo AI-C111 RFLP frequencies, these Xl and X2 frequencies were typical of previously sampled Caucasian populations [33].

In contrast to apo AI-C111 RFLPs, the high level of polymorphism for the X&T RFLP al- lowed for investigation of a ‘protective’ apo B genotype by classification of nonagenarians among each of the genotypes (7 subjects were X1X1 homozygotes, 9 were X1X2 heterozygotes, and 5 were X2X2 homozygotes). Genotype-specific differences in serum levels of lipids, lipoproteins, and apolipoproteins that are known ‘risk’ factors for atherosclerosis were evaluated. Table 4 illustrates results from this statistical analysis using general linear model procedures to compare mean values for these measures among apo B XbaI genotypes. Although the small numbers of nonagenarians in each genotypic class limits our power to detect significant phenotypic effects, this preliminary analysis did not reveal any significant differences among these groups. A trend toward significance was detected in the relationship between the apo B XbaI genotypes and the LDL-C/apoB ratio. The

absence of differences in ‘risk’ factors among apo B XbaI genotypes may explain the failure to identify a ‘protective’ apo B genotype that dis- tinguishes nonagenarians.

To further assess the potential phenotypic effects of apo B genotypes, nonagenarians were divided according to phenotypes, and evaluated for differences in distribution of genotypes among particular phenotypic classes using general linear model procedures and &i-square tests. The distribution of genotypes was random and comparable between subsets for serum cholesterol categoriza- tion according to the National Cholesterol Educa- tion Program (desirable levels < 5.17 mmol/l, borderline high levels of 5.17-6.20 mmol/l, and high levels 2 6.21 mmol/l). Similarly, no genotypic differences were detected among groups divided by LDL-cholesterol concentration (< 3.36 mmol/l, 3.36-4.11 mmol/l, 4.11-4.90 mmol/l, 2 4.91mmol/l) and HDL-cholesterol concentra- tions (< 0.91 mmol/l, 0.91-1.41 mmol/l, 2 1.42 mmol/l).

Discussion

Our present effort to characterize the clinical and lipid phenotype of very long survivors and to relate these findings to apolipoprotein genotypes has provided further insight into the complexity of longevity. While lipid, lipoprotein and apolipoprotein profiles (Table 2) of the nonagenarians studied are generally favorable by current standards for younger individuals, broad diversity of lipid characteristics persists into the tenth decade of life. Thus, both lipid phenotype, body habitus, cardiovascular symptoms and therapy, and dietary practices are randomly distributed in this age group. Of particular note, the putative low risk profile with a low total blood cholesterol, a low LDL-cholesterol, low blood triglycerides, and apo B levels, and/or high HDL-cholesterol and apo AI levels, are not necessarily found in the long survivor. Moreover, the phenotypes are not de- monstrably concordant with any particular dietary pattern insofar as total calories, carbohydrate fat or protein are concerned.

The interesting persistence of a significantly greater HDL-cholesterol in women as compared to men may be subtly important in the overall

superior survival in females. However, no patient in this entire series demonstrated a markedly elevated HDL-cholesterol (hyperalphalipoprotein- emia). Considering that the mean HDL-cholesterol level is almost unchangeable across the adult lifespan, the absence of extremely high HDL- cholesterol levels in our elderly group is not suffi- ciently explained on a senescent inability of gut and liver synthesis of HDL-cholesterol or apo AI.

To determine if nonagenarians possess distinctive ‘protective’ genotypes that may explain their longevity. subjects have been typed with respect to RFLPs in genes involved in cholesterol metabolism. The allele frequencies for MspI, PstI, and SstI RFLPs in the apo AI-C111 gene cluster were typical for those from larger population studies, and did not distinguish the nonagenarians from other Caucasian populations. Similarly, allele frequencies for the apo B XbaI RFLP were similar to those from larger studies of Caucasian populations. To investigate the absence of ‘protective’ apo B XbaI genotypes in the nonagenarians, genotypic effects on serum levels of lipids, lipoproteins, and apolipoproteins that are known ‘risk’ factors of atherosclerosis were evaluated. Al- though rather small sample numbers limited statistical power to detect such effects, these preliminary studies failed to identify any significant genotypic effects on these phenotypes. Only a trend toward significant differences in LDL- C/ape B ratios across XbaI genotypes was observed, a reflection of heterogeneity of apo B-containing lipoproteins [34]. Our results are similar to those from other studies that have been unsuccess- ful in detecting phenotypic effects of the apo B XbaI RFLP, as contrasted to studies that detected significant effects on ‘risk’ factors and heart disease (for review, see refs. 10, 11). Our failure to identify ‘protective’ genotypes may result from the absence of genotypic effects in this population, such that possession of specific apo B XbaI alleles does not alter longevity.

Acknowledgements

The authors would like to acknowledge the support of the American Heart Association Nebraska Affiliate and the American Aging As- sociation. Also, the assistance provided by Joseph

145

E. O’Brien, M.D. (MetPath Laboratories) and by the staff in the Bloomfield Clinic was invaluable in this study. The authors thank Drs. Jan Breslow and James Scott for kindly providing cDNA probes, and Laura Cox for excellent technical assistance.

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Lipid phenotypes, apolipoprotein genotypes and cardiovascular risk in nonagenarians