Mevalonic acid MVA, an intermediate in choles- terol synthesis and the immediate product of HMG
CoA reductase is present in plasma and urine [5]. Previous studies have shown that the concentrations of
mevalonate in plasma and urine parallel the rates of hepatic and whole body cholesterol synthesis in both
animals and man [6,7]. There is also a very good correlation between plasma MVA and the rate of incor-
poration of deuterium into plasma free cholesterol in humans [8]. Plasma MVA concentrations exhibit diur-
nal variations, whereas the 24-h urinary MVA excretion reflects the integrated plasma concentrations and thus
provides a more practical way of measuring the influ- ence of dietary or pharmaceutical manipulations on
cholesterol synthesis than conventional sterol balance techniques [9].
In the present study, we evaluated the effects of high doses of atorvastatin 40 – 160 mgday on steady state
concentrations of plasma lipids and lipoproteins, plasma MVA and the 24-h urinary excretion of MVA
in patients with well characterized HoFH.
2. Methods
2
.
1
. Patients Patients with HoFH attending the lipid clinics at the
University of the Witwatersrand and University of Cape Town participated in the study. The diagnosis of
HoFH was based on the presence of an untreated serum LDL cholesterol consistently greater than 12
mmoll; the appearance of xanthomas in the first decade of life; documentation in both parents of hyper-
cholesterolaemia or clinical features of heterozygous FH; and confirmation by DNA analysis for LDL recep-
tor gene mutations. Familial defective apo B-100 due to a glutamine to arginine substitution at codon 3500 of
the apo B gene was excluded by DNA analysis in all subjects. Patients were excluded from the study if they
had hepatic or renal dysfunction, or had known hyper- sensitivity to HMG CoA reductase inhibitors. No pa-
tient was receiving LDL-apheresis at the time of the study. All recruited patients gave their written informed
consent to the study which was approved by the Re- search Ethics Committees of both Universities.
2
.
2
. Trial design The study was a double-centre, dose escalation study.
After an untreated baseline fasting lipid profile, patients received atorvastatin at doses of 40, 80, 120 and 160
mgday with a minimum period of 4 weeks of therapy between dosage escalation. Initially, all patients re-
ceived 40 and then 80 mg atorvastatinday. In patients who did not achieve an LDL cholesterol goal of 5 3
mmoll, the dose of atorvastatin was increased further to 120 mg and subsequently to 160 mgday, with the
limitation that in all patients the maximum atorvastatin dose did not exceed 2.5 mgkg body wt per day. All
patients were counselled on a standard low-cholesterol, low-saturated fat diet and the diet was reinforced at
each visit to avoid the confounding effect of diet on lipid levels during the study. Venous blood samples for
lipoprotein analysis were drawn in the morning after an overnight fast of at least 10 h. Samples were obtained at
baseline and then at the time of each dose escalation. Interim biochemical safety tests were also regularly
performed.
2
.
3
. DNA analysis DNA screening for three locally prevalent founder-
related Afrikaner LDL receptor LDLR mutations, D206E Afrik-1, V408M Afrik-2 and D154N Afrik-
3, was performed in a single reaction by a multiplex amplification refractory mutation system-polymerase
chain reaction [10]. The DNA samples were also screened for mutation P664L FH-Gujerat previously
identified in South African Indians [11]. After screening for familial defective apolipoprotein B-100, subjects
negative for these mutations underwent a more exten- sive search by heteroduplex andor single-strand con-
formational polymorphism analysis [10,12].
2
.
4
. Measurement of me6alonic acid Venous blood samples as well as 24-h urine speci-
mens were collected for measurement of the plasma and urine concentrations of MVA. Three separate 24-h
urine samples were collected before the initiation of atorvastatin therapy and then one after at least 4 weeks
on each dose of atorvastatin. Urine and plasma samples were stored at − 70°C and, after thawing, were ana-
lyzed by radioenzymatic methods as previously de- scribed [13].
2
.
5
. Other measurements Total cholesterol, HDL cholesterol and triglycerides
were analyzed by enzymatic methods using automated techniques. The LDL cholesterol was calculated using
the Friedewald formula [14]. Biochemical safety tests alanine transaminase, aspartate transaminase and cre-
atine kinase were also performed at regular intervals throughout the study. Trough predose plasma ator-
vastatin concentrations were measured by high-perfor- mance
liquid chromatography
tandem mass
spectrometry immediately prior to each atorvastatin dose escalation [15]. The effect of increasing atorvas-
tatin dosage on plasma atorvastatin concentrations was expressed as the change in concentration calculated
as follows:
change = [trough concentration after dose in- crease − trough concentration before dose increase
trough concentration before dose increase] × 100
2
.
6
. Statistical analysis Comparisons were performed using one-way analysis
of variance ANOVA for repeated measures followed by student Newman – Keuls method for post hoc com-
parisons using Sigmastat Statistical Software for Win- dows Jandel, CA. The relationship between the
changes in MVA and LDL cholesterol was explored by univariate regression analysis using JMP Statistical
Software Package Version 3.1 SAS Institute. In all comparisons, P B 0.05 was considered significant. Re-
sults are expressed as mean 9 S.E.M.
3. Results
