University Of San Francisco Researcher Presents Results Of An Investigation Of 18 Hyperlipidemic Children In The Bay Area

February 22, 2002 – San Francisco -- Paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL). The antioxidant properties of HDL are due largely to the ability of its associated PON1 to both remove lipid peroxides from low-density lipoproteins (LDL) and  prevent the transformation of LDL into oxidized, atherogenic particles (OxLDL). 

PON1 is named for its ability hydrolyze a synthetic compound  known as “paraoxon.” Numerous genetic polymorphism have been identified within the PON1 locus that affect this function.

Perhaps the strongest is a single amino acid substitution that occurs at position 192. A glutamine (Q) at PON1 192 is associated with diminished enzymatic activity. At the same time, an arginine (R) in the same position results in a high-activity isoform. A second polymorphism of the PON1 gene results in an amino acid substitution at position 55. A smaller effect on activity against paraoxon has been noted at this locus with a leucine (L) at aa 55 being associated with greater activity than a methionine (M).

Increased PON1 isoenzymatic activity against paraoxon is opposite to its specificity and activity with respect to the hydrolysis of lipid peroxides. In fact, increased atherosclerosis has been associated with the “high paraoxon hydrolysis activity” PON1 R192 and L55 alleles. However, conflicting reports describe significantly diminished PON1 activity in patients with acute myocardial infarction (i.e., heart attack) and an increased risk of atherosclerotic symptoms in individuals with the low paraoxon activity PON1 192QQ genotype. Substantial inter-individual variation in PON1 activity has been described that may be independent of both the 55 and 192 polymorphisms.

While adult studies have demonstrated genetic regulation of PON1 activity, no investigations had been undertaken within the pediatric population. Accordingly, Michele Mietus-Snyder, MD of the Departments of Physiological Nursing and Pediatrics at the University of California at San Francisco (UCSF) has evaluated PON1 activity in children and adolescents with high lipid levels. Her study, conducted over a six-month interval, closely examined l8 pre-screened volunteers enrolled in an ongoing nutritional intervention in pediatric hereditary hyperlipidemia.

Dr. Mietus-Snyder will release her findings in detail during her presentation at the spring meeting of the American Physiological Society (APS), being held in San Francisco, CA February 20-23, 2002.

The Study

The UCSF study team conducted a randomized, placebo-controlled, double-blinded crossover study of l8 children with hereditary hyperlipidemia. None of the participants were on lipid-lowering or other medications and none were smokers.  Familial hypercholesterolemia (FH) was defined as both low-density lipoprotein (LDL) cholesterol above 130 mg/dl and a parent with the disorder. Familial combined hyperlipidemia (FCH) was diagnosed as either LDL cholesterol in excess of 130 mg/dl, a triglyceride count of greater than 150 md/dl, or both and a parent with any of these three phenotypes.  12 study participants carried the diagnosis of FCH; 6 of FH.

A nutritional education program was used for the six month study period. After six weeks of diet alone the participants were randomized to receive antioxidant vitamins C and E or placebo. This was followed by a six-week washout and a six week crossover phase. At baseline and at each six-week interval, three-day food records were obtained and lipoprotein profiles were measured.  Serum was assayed for PON1 activity and DNA was isolated for PON1 genotyping.

PCR primers previously characterized for amplification of the PON1 gene polymorphism were synthesized and amplifications were performed.  Plasma PON1 activity was determined by measuring the hydrolysis of paraoxon using an adaptation of spectrophotometric methods.

Results

Baseline PON1 Activities by Genotype

The lowest tercile of activity was seen in seven participants who had a QQ genotype at position 192.  Moderate activity was observed in the eight participants with a QR genotype. The highest activity was found in the three individuals with an RR genotype.  Although a tendency towards lower levels existed when an M allele was present at position 55 in those individuals who had either a QQ or QR genotype at position 192, these differences were not significant.  Any additional effect of the M allele on the RR genotype could not be assessed, as all RR participants were also homozygous for the L allele. 

PON1 Genotype Frequencies at Condons 192 and 55

The gene counting method showed that allele frequency at codon 192 was Q=0.61 and R=0.39. and at codon 55 it was L=0.81 and M=0.19.

PON1 Activities With Nutritional Intervention

PON1 activity decreased slightly in the first six weeks of nutritional intervention among all three of the PON1 192 genotype groups. These lower levels remained for the PON1 192QQ group but gradually drifted back up within the other subjects by the washout period (l8 weeks). This trend coincided with a modest reduction in HDL triglycerides and in the reported total, saturated, monounsaturated and polyunsaturated dietary fat intake. The mean HDL cholesterol levels climbed during the same interval but remained low. Interestingly, dietary fat intake also climbed again toward the end of the study. None of these trends reached levels of significance in this small study population, nor did PON1 activity vary significantly after a six week course of Vitamins C and E. 

Conclusions

Based on these findings Dr. Mietus-Snyder concludes that: 

• The results from this first-of-its-kind investigation examining the effects of PON1 polymorphisms in hyperlipidemic children are consistent with findings reported from similar studies in adults. 

• Although a decrease in PON activity accompanied the reduction in fat intake achieved with nutritional counseling, no significant trend was observed.  This finding and related independent research has however led to the speculation that specific fatty acids might play a regulatory role in PON1 expression and therefore impact HDL antioxidant activity.

• HDL cholesterol levels increased slightly over the six month study period while PON1 activity decreased.  HDL triglyceride levels decreased – coincident with diminished PON1 activity.  This suggests that a change in HDL speciation may be occurring with the dietary intervention that could affect the population of PON1-carrying HDL particles.

• Despite changes in HDL cholesterol and triglyceride levels, the mean HDL levels remained low throughout the study. This may reflect the fact that two-thirds of the study participants had FCH with elevated fasting TG levels and concomitant low HDL. Their low HDL levels may have also reflected their dietary fat restriction as the majority of the subjects were on were on a relatively low fat diets at baseline.

Dr. Mietus-Snyder believes that these discoveries pose new and important questions about the regulation of PON1 activity.  Additional research will be necessary to unravel the important genetic-environmental interactions that may affect PON1 gene expression and the risk for atherosclerosis, particularly as they relate to children. 

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The American Physiological Society (APS) was founded in 1887 to foster basic and applied science, much of it relating to human health. The Bethesda, MD-based Society is one of the world’s most prestigious organizations, totaling more than 10,000 members, including physiological researchers and academicians. Clinical physiologists investigate the function of the human body, including the effects of genes, diseases, exercise and metabolism. Their primary professional commitment is to understand these processes and functions so that promising new cures can be developed.  The APS publishes 3,800 articles in its 14 peer-reviewed journals every year. Physiological Genomics, the newest APS journal and the conference co-sponsor, is among the Society’s flagship publications.

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