¹Division of Endocrinology, Metabolism and Diabetes, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver, CO 80262

²Hoffmann-LaRoche Inc., 340 Kingsland Street, Nutley, NJ 07110-1199, USA

Correspondence to: R H Eckel, Campus Box B151, UCHSC, 4200 E. 9th Ave, Denver, CO 80262, USA. Robert.Eckel@UCHSC.edu.

OBJECTIVES: After 10 d of orlistat administration (120 mg three times/day), the primary objective was to determine the drug's effect on postprandial plasma lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities on day 10 after an oral fat-load. The secondary objectives were to determine the effects of orlistat on 12 h postprandial measures of: (1) preheparin HTGL and LPL; and (2) serum triglycerides, very-low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and free fatty acids.

METHODS: Twenty-four normal-weight, healthy male volunteers were randomized to either 120 mg orlistat (n=12) or placebo (n=12) three times a day with meals for 10 d. Preheparin LPL and HTGL activities and LPL specific activity were measured in the fasted state on days 1, 5, and 10. On days 5 and 10 the study medication (orlistat or placebo) was taken at the beginning of a fat-rich breakfast and serum lipid and lipoprotein levels monitored for 12 h postprandially. On day 10, 15 min postheparin HTGL activity was measured 8 h after the fat-rich breakfast.

RESULTS: No differences were found between groups in fasting levels of preheparin LPL or HTGL activity or in LPL-specific activity on days 1, 5 and 10. No difference was found between the two treatment groups in postheparin HTGL activity 8 h after the fat-rich breakfast. Also, no differences were found between the two groups in plasma triglycerides or lipoproteins.

CONCLUSION: The results indicate that the oral administration of orlistat (120 mg t.i.d.) does not significantly alter plasma triglycerides or lipoproteins, and that the inhibitory effect of orlistat on lipases is limited to the gastrointestinal tract and is not manifested systemically.

International Journal of Obesity (2000) 24, 187-194

orlistat; tetrahydrolipstatin; lipoprotein lipase; hepatic triglyceride lipase; lipoproteins

Introduction

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) are endothelial lipases that regulate lipoprotein transport in humans. LPL hydrolyzes the triacylglycerol core of large, triglyceride-rich lipoproteins, specifically chylomicrons and very low density lipoproteins (VLDL), whereas HTGL also acts on smaller and more dense particles including intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Both triacylglycerol hydrolyase and phospholipase activities have also been described for HTGL. LPL and HTGL exhibit strong base-pair sequence similarities to pancreatic lipase (PL) in the region of the gene which codes for the active site. Since the residues at the active site are conserved in all known lipoprotein lipases, it is likely that HTGL is similar to LPL and PL. Orlistat is a new anti-obesity drug of the compound tetrahydrolipstatin (THL) that limits fat absorption by the inhibition of PL activity. In vitro experiments demonstrate that orlistat has an IC₅₀ of 122 ng/ml for PL from human duodenal juice, but an IC₅₀ of about 300 ng/ml for LPL.¹ If significant systematic absorption of orlistat occurs after oral administration, the drug may inhibit LPL and HTGL activities and thus may also alter normal lipoprotein metabolism. Of note is the fact that orlistat given to rats at a dosage of 500 and 1000 mg/kg/d resulted in hypertriglyceridemia.²

Because orlistat may inhibit systemic plasma lipases as well as gastrointestinal lipases, the effect of orlistat on LPL and HTGL activity when serum triglycerides increase postprandially needs to be established. Therefore, the effects of orlistat on postprandial pre- and postheparin LPL and HTGL activities and on postprandial plasma lipoprotein concentrations were examined in this clinical trial. The study was designed to descriptively compare two treatments, oral orlistat vs oral placebo administration, on plasma pre- and postheparin lipases and plasma lipoproteins.

The traditional method for measuring LPL and HTGL enzymatic activities relies on the intravenous administration of heparin, which releases the lipases into the circulation. Although useful, this approach is non-physiologic and cannot assess the impact of short perturbations, e.g. the effect of an acute meal or drug, on lipase activities. Thus, a procedure to measure plasma lipolytic activities without prior administration of heparin was developed.³ With this method, basal or 'preheparin' total plasma lipolytic activity (PLA) is measured directly, as is the activity of HTGL. LPL activity then is determined as the difference between PLA and HTGL activity. In preheparin plasma samples, basal (fasting) levels of LPL activity tend to be more variable than basal HTGL levels.

Although the relative contribution of HTGL and LPL to the totally lipolytic pool is similar in both pre- and postheparin plasma,³ two distinct techniques are employed for measuring systemic lipase activity. The assay of preheparin plasma quantifies small amounts of circulating enzyme which are bound to lipoproteins.³ In contrast, the displacement of lipases by heparin from endothelial binding sites yields much larger amounts of enzyme, representing what is though to be the total physiologically available lipase pool. With the 'no heparin' technique, serial measures may be obtained without the confounding features of heparin-induced anticoagulation, depletion of tissue-bound lipases with the corresponding delay for restoration, and the substantial lipolysis following heparin adminstration which causes perturbation of plasma lipid levels.

Methods and procedures

Study design

The study was conducted in full conformance with the principles of the Declaration of Helsinki (as amended in Tokyo, Venice and Hong Kong). The protocol was approved by the Colorado Multiple Institutional Review Board of the University of Colorado Health Sciences Center (UCHSC), and by the UCHSC General Clinical Research Center (GCRC) Scientific Advisory Committee. The aims, methods, objectives and potential hazards of the study were fully explained to the subjects, and written informed consent was obtained prior to the screening visit. Every subject had the right to withdraw from the study at any time and for any reason.

This was a double-blind, randomized, parallel group, placebo-controlled study of 24 healthy adult male volunteers, age range 22-55 y. The volunteers were screened in the outpatient clinic of the GCRC a maximum of 14 d prior to study initiation. Screening included a history and physical exam, electrocardiogram, vital signs (blood pressure, heart rate, respirations) and fasting blood work. Laboratory determinations were done for the following parameters: complete blood count (CBC), activated partial thromboplastin time (to confirm normal coagulation status), liver function tests (SGOT, SGPT, alkaline phosphatase), and routine chemistries, including albumin, glucose, creatinine and total protein. A routine urinalysis was done, as well as a urine drug screen for cocaine and cannabinoids. None of the subjects were taking any medications. Because intravenous heparin was to be administered on day 10 of the study, each subject was advised to refrain from taking any aspirin- or ibuprofen-related products within 1 week before and/or during the 10-d study.

Upon verification of normal screening test results, each individual was assigned a subject number which automatically entered him into one of the two arms of the study (orlistat or placebo) per double-blinded design. The subject randomization numbers were allocated sequentially in the order in which the subjects were enrolled.

Each subject arrived on the GCRC of UCHSC on the morning of study day 1, after having fasted (water only) for 12 h overnight. Blood samples for preheparin plasma LPL and HTGL activity (and LPL specific activity), triglycerides (TG), VLDL-cholesterol (VLDL-C), HDL-cholesterol (HCL-C), LDL-cholesterol (LDL-C) and free fatty acids (FFA) determinations were drawn in the fasted state prior to breakfast. The study subject then received orlistat 120 mg capsules or placebo capsules (per randomization schedule) three times per day (t.i.d.) with meals on days 1-4 of the study, without any specific dietary prescription or restrictions. At each time of administration, the capsule was taken with 8 oz of water. Orlistat or placebo was supplied as blister cards; each card contained three capsules for consumption during each day except days 5 and 10 of the trial. Treatment medication for the whole study was dispensed to the subject on day 1. The dose of 120 mg orlistat t.i.d. was chosen in order to observe the effect of treatment with the established therapeutic dose⁴ on LPL and HTGL activity at a time when steady-state plasma concentrations would be expected.

On the morning of day 5 after a 12 h overnight fast, the study subject was required to eat a prescribed fat-rich breakfast and take a single dose of his assigned medication. Each subject ingested identical foodstuffs for the prescribed meal. The fat-rich breakfast (composition: ~830 kcal, ~28 g fat) consisted of two eggs fried in 1 tsp butter, one strip of bacon, two slices of white toast with 1 tsp butter, one raisin bran bagel or raisin bran muffin, 6 oz low-fat milk and 6 oz fruit juice.

Blood samples for plasma preheparin LPL and HTGL levels and lipid profile determinations were drawn in the fasted state prior to breakfast, and then hourly over the 12 h postprandial period. The subject was not given lunch on this day, and thus the mid-day dose of study medication on day 5 was skipped. Orlistat or placebo treatment resumed with the evening meal on day 5 after the 12 h postprandial blood samples had been drawn. Pharmacokinetic samples for orlistat were drawn on day 5 in the fasted state, and then at the 2, 4, 6, 8, 10 and 12 h timepoints along with the other postprandial blood draws. The subject continued his assigned study medication t.i.d. with meals on days 6-9, again without dietary prescription or restrictions.

On the morning of day 10 after a 12 h overnight fast, blood was again drawn for preheparin levels of LPL and HTGL and orlistat pharmacokinetics. The subject then received orlistat or placebo with a fat-rich breakfast identical in form and composition to the one consumed on day 5. However, on this day (unlike day 5), 7 3/4 h after ingestion of the morning dose of study medication, the subject was given an intravenous bolus of heparin sodium (100 IU/kg). Exactly 15 min after the heparin bolus was given, blood samples were drawn for measurement of postheparin plasma LPL and HTGL activities and lipid profile. At this time labs for CBC, chemistries and UA (as done at baseline) were also repeated. The subject was subsequently monitored on the GCRC for 4 h for any possible secondary complications from the heparin injection. An exit physical exam (including vital signs) was then done and the subject was discharged home after condition stability had been established and documented. Upon discharge, subjects were cautioned to avoid use of asprin- or ibuprofen-containing products for approximately 20 d.

Analytical methods

All samples for pharmacokinetic analysis were stored until the randomization code was broken. Only samples from subjects randomized to active treatment (120 mg orlistat t.i.d.) were assayed for plasma concentrations of unchanged orlistat. The analytical method (LC/MS/MS) used to measure plasma concentrations of orlistat (Ro 18-0647) had a limit of quantification of 0.2 ng/ml using 1 ml of plasma aliquot.

Assay of lipase activities

Assays of total plasma lipolytic activity for measurements of LPL and HTGL were performed after 0.5 ml of plasma had been placed over a 0.5 ml volume column of heparin-Sepharose 6B (Pharmacia, Piscataway, NJ) that had been previously equilibrated in 0.02 M barbital sodium, 0.3 M NaCl buffer, pH 7.4, at 4°C.⁵ After washing the column three times with 0.5 ml of the buffer, enzyme activity was eluted with 1.5 ml of barbital sodium/NaCl buffer containing 6 mg/ml heparin sodium (Fisher, Fair Lawn, NJ). Postheparin plasma was not put onto the column, but was diluted 1:25 prior to assay.

For each lipase assay, two different substrates were used: one for the specific measurement of total lipase activity and one for HTGL activity. New substrates were prepared each assay day. For total preheparin lipolytic activity (PLA), the substrate was prepared with 10 mg triolein (Sigma, St Louis, MO), 8 Ci [1-¹⁴C]triolein (Amersham, Arlington Heights, IL), and 0.48 mg egg phosphatidylcholine (Calbiochem-Behring, La Jolla, CA). After drying under nitrogen, lipid components were emulsified in a 4 ml mixture of 10% fatty acid-poor bovine serum albumin (Miles, West Haven, CT), pooled normal human serum, 2 M Tris buffer (pH 8.2), and distilled water (0.8:1.3:1.0:0.9) by 100 s of sonication (10 s on followed by 10 s off for 10 cycles; Model W-220F, Heat Systems-Ultrasonics, Plainview, NY) at 4°C. For HTGL the substrate volume was altered by the addition of NaCl to a final concentration of 3.89 M, and the final pH was adjusted to 8.6 with 2 M Tris-HCl. Serum was omitted and substrate volume was maintained with water.

After heparin elution from heparin-Sepharose 6B columns, 0.15 ml aliquots of enzyme eluate were incubated with 0.05 ml of each substrate. Before addition of the enzyme, the substrates were preincubated for 90 min at 37°C. The reaction was carried out at 37°C and terminated after 90 min with a fatty acid extraction mixture. Reaction vessels were shaken for 5 min on a shaker (Eberbach, Ann Arbor, MI) and centrifuged at 600 g for 20 min. A 0.5 ml aliquot of the upper phase was removed and counted in a scintillation counter (Searle Mark III, Des Plains, IL).

After correction for recovery, results were expressed as nmol FFA liberated over 1 h per ml of plasma. Preheparin LPL activity was calculated as the difference between the amount of total lipase activity measured using the serum-containing substrate and that amount measured as preheparin HTGL. Post-heparin LPL was calculated as the difference between total postheparin lipolytic activity and postheparin HTGL.

Using this methodology, Glaser et al⁵ demonstrated an interassay coefficient of variaion of 10.5% for total lipase activity and 11.4% for HTGL. Intra-assay variation was 3.3±0.3% for total lipase activity, and 4.8±0.5% for HTGL activity. The sensitivity of the assay was 0.1 nmol FFA/ml/h.

Assay of LPL mass

The lipoprotein lipase ELISA is a sandwich ELISA utilizing an affinity purified primary antibody and a secondary biotinylated antibody.⁶ For maximum sensitivity, each of the following steps was done after overnight storage of plasma at 4°C. Immulon-4 plates were coated with 100 l of an affinity-purified chicken LPL antibody diluted (1:120) in NaHCO₃ (pH=9.5). The plates were washed and blocked with a solution (150 l) containing Bovine Serum Albumin (BSA) (0.1%) and Triton X-100 (0.01%). Pre- and postheparin plasma (100 l) diluted 1:2 in NaCl (2 M), phosphate buffered saline (PBS) (2´, pH=7.4), BSA (0.2%) and Triton X-100 (0.2%) with a cocktail of protease inhibitors (aprotinin, benzamidine, PMSF and EDTA) was added to the plate. Purified bovine milk LPL was used as a standard curve (100-0.78 ng/ml) along with a quality control of bovine LPL. After washing, a biotinylated secondary lipoprotein lipase antibody was diluted (1:3000) and added to the plate. Streptavidin conjugated HRP (1:4000) was then added followed by a color reaction using o-phenylinediamine. Plates were read at 492 nm in a Dynatech ELISA reader and LPL mass quantified based upon a log-logit transformed standard curve. The sensitivity of the assay was 1 ng/ml. LPL specific activity (nmol FFA/h/ng LPL) was defined as LPL activity (nmol FFA/mL/h) divided by LPL mass (ng LPL/ml).

Assays of lipoproteins, lipids and FFA

Serum cholesterol was measured with a modification of the Trinder method.⁷ LDL and VLDL particles were precipitated and removed from serum by ultracentrifugation,⁸ and the cholesterol in each density fraction measured by the cholesterol method.⁷ The HDL cholesterol remaining in the supernatant was also measured with the cholesterol method.⁷ Serum triglycerides were hydrolyzed by lipase and the liberated glycerol was measured with colorimetric reaction as described by Kohlmeier.⁹ Serum FFA levels were measured enzymatically with a colorimetric end point.¹⁰

Data evaluation and statistical analysis

The alternative hypothesis specified in the protocol was:

|mean postheparin HTGL activity (orlistat)

-mean

postheparin HTGL activity (placebo)|

<0.25 mean

postheparin HTGL activity (placebo)

This equation is mathematically equivalent to:

the mean postheparin HTGL activity (orlistat)/

mean postheparin HTGL activity (placebo)

falling inside the range of 0.75-1.25.

Therefore, to test equivalence, a 90% confidence interval was computed around the ratio of the two treatment groups with respect to postheparin HTGL activity. Another way of stating the alternative hypothesis is that the orlistat mean falls within the interval of the placebo mean±25%.

Calculations of area under the effect curve (AUEC) were estimated by the trapezoidal rule, and were baseline corrected. Student's t-test (unpaired) was used to test for significant differences (P<0.05) between the VLDL-C, LDL-C, HDL-C, FFA, TG, HTGL, LPL and LPL specific activity AUEC of the two treatment groups, and also to test for differences between the two groups with respect to the individual timepoint values for HTGL, LPL and LPL specific activity. An approximate 90% confidence interval around the ratio of the two treatment group HTGL values was used to test for equivalence between them.

Results

Study population

After the randomization code was broken at the end of the study, it was found that the placebo group included subjects with ages ranging from 22 to 55 y (mean±s.e.m., 31±2 y), and the age range of the orlistat group was 24-47 y (35±2 y). There was no difference in age between the two groups (P=0.21), and no difference in height (placebo vs orlistat, 178±2 vs 179±1 cm, P=0.69). Body weights did not differ between the groups (placebo vs orlistat, 79.6±2.6 vs 86.0±3.3 kg, P=0.14), nor did body mass index (placebo vs orlistat, 25.1±0.5 vs 26.8±1.0 kg/m², P=0.14).

Pharmacokinetic results

Plasma samples collected during day 5 were analyzed for the presence of unchanged orlistat. Mean plasma concentrations of orlistat in the range of 0.20-3.37 ng/ml were detected in the plasma of about 66% of the 2 h samples, in the range of 0.27-2.07 ng/ml in about 66% of the 4 h samples, and in the range of 0.32-0.54 ng/ml in about 33% of the 6 h samples. Orlistat was not detected in plasma samples at 8, 10 and 12 h.

Pharmacodynamic results

There were no differences between groups in pre-heparin LPL or HTGL activity or in LPL specific activity measured in the fasted state on days 1, 5 and 10 (Table 1). As shown in Table 2, on day 5 there were no differences between the placebo group and the orlistat group in AUEC over the 12 h postprandial period for preheparin HTGL, LPL or LPL specific activity, nor for any of the plasma lipoproteins.

On day 5, for 12 h following the challenge of the fat-rich breakfast with orlistat or placebo administration, no differences between the two treatment groups were evident with respect to preheparin HTGL activity (Figure 1A), LPL activity (Figure 1B), or LPL specific activity (Figure 1C) when analyzed by specific timepoint comparisons. Postprandial preheparin LPL activity and LPL specific activity tended to be lower at 8 h and 9 h after orlistat treatment compared to placebo treatment, but the differences did not reach statistical significance (LPL at 8 and 9 h, P=0.18 and 0.09, respectively; LPL specific activity at 8 and 9 h, P=0.18 and 0.27, respectively). When the data in Figure 1A-C were analyzed as AUEC, there were no differences between the two treatments in the calculated areas of any of the three enzymatic variables (Table 2).

The lipid and lipoprotein profiles over the 12 h postprandial period on day 5 were similar in the two treatment groups, as illustrated in Figure 2A-E. The postprandial excursions of the lipoproteins seemed to follow a typical pattern, and corroborated the analyses of the lipase activities.

Day 10 included the administration of a bolus of intravenous heparin 7 3/4 h after ingestion of a fat-rich meal plus orlistat or placebo. The measurements of postheparin HTGL and LPL activities and postheparin LPL specific activity were not different between the two treatment groups (Table 3).

Adverse events

Overall, orlistat was well tolerated by the subjects. All reported adverse events were associated with the lower gastrointestinal tract. Eleven subjects receiving orlistat reported gastrointesinal adverse events assessed as 'probably related' to the study medication. One case of liquid stools and one case of abdominal distention, both reported by subjects receiving orlistat, were assessed as being of severe and moderate intensity, respectively. These symptoms were self-limiting, and did not negatively affect subject compliance. The three adverse events (soft stool, abdominal pain, oily stool) reported by two patients receiving placebo were assessed as being unrelated to the study medication.

Discussion

Pharmacokinetics

It has been shown in previous studies that orlistat acts locally in the gastrointestinal tract to inhibit pancreatic and gastric lipases.¹¹ Results of Phase 1 plasma monitoring studies¹² confirmed that the systemic exposure of orlistat is extremely low and no pharmacokinetics can be defined. On the other hand, for a locally acting therapeutic product, a potential risk exists if the active substance is absorbed. This study was carried out to ensure that the systemic exposure from oral orlistat administration was not different than documented in previous clinical studies.

Detectable orlistat plasma levels (0.2-3.37 ng/ml) were most frequently found 2-6 h after the morning dose on day 5. The detected levels of drug did not exceed those observed in the plasma monitoring program¹² at the same 120 mg dosage level (<5 ng/ml).

Pharmacodynamics

In the present study it is apparent that oral orlistat, as administered, had no effect on lipoprotein metabolism in normal male subjects. The postprandial lipid and lipoprotein responses were examined on day 5 (after 4 days of orlistat 120 mg t.i.d.) when a single a.m. dose of orlistat was administered within 5 min of meal consumption. There were no treatment differences in the AUEC for any of the postprandial lipids or lipoproteins, including TG, FFA, HDL-C, LDL-C and VLCL-C. Also, there was no difference in the peak triglyceride response between the orlistat-treated subjects and the placebo-treated subjects. Results from a multi-center clinical trial¹³ showed that orlistat (120 mg t.i.d. for 4 weeks) was associated with a significant decrease in fasting serum LDL-C, but no change in fasting serum triglycerides.

In contrast to these results, Reitsma et al¹⁴ found significant (P<0.05) differences in the 8 h postprandial plasma TG concentrations (AUC) between untreated subjects and tetrahydrolipstatin (THL)-treated subjects after an oral fat-load (40% fat), with the TG AUC being less in the THL-treated group. However, there were several differences between the study reported here and the study by Reitsma et al.¹⁴ First, the subjects studied by Reitsma et al were all hyperlipidemic with fasting plasma total cholesterol levels of at least 6.2 mmol/l and LDL-C levels of at least 4.2 mmol/l, and were therefore not representative of subjects with normal lipid metabolism. Second, the subjects received THL treatment and were on a prescribed low-fat, low-cholesterol (AHA Step 1) diet for a total of 8 weeks prior to the oral fat-load challenge. In the Reitsma paper, it is stated that 'fasting plasma TG and HDL cholesterol levels were unchanged after therapy, suggesting that the hepatic production of TG-rich particles and clearance by lipoprotein lipase had not been affected by THL.'¹⁴ These factors represent major differences in study design when compared to the present study.

The pharmacodynamic results from this study demonstrate that postheparin HTGL activity was similar in both treatment groups. The hepatic enzyme, which accounts for approximately 60% of the total plasma lipolytic activity, was not affected by the prescribed multiple dose regimen of 120 mg orlistat t.i.d. It is also evident from the data shown here that orlistat treatment for 10 d did not alter fasting levels of preheparin HTGL and LPL or LPL specific activity. The activity of the enzymes remained within a narrow index over the course of the study. Changes were of the same magnitude and measures had the same variability in placebo and orlistat groups.

There was a tendency for LPL activity and LPL specific activity to decrease at the 8 h and 9 h timepoints during the postprandial period on day 5, but the concentration of orlistat, the only inhibitor of the enzyme, was zero in these instances. Therefore, the 2 h period during which postprandial LPL activity tended to be lower apparently represents normal fluctuation in this highly variable metabolic enzyme. However, it is also important to note that LPL in both pre- and postheparin plasma is calculated by subtraction of HTGL from total lipase activity. This method of LPL determination, particular when the LPL/ HTGL ratio is low (that is in postheparin plasma), makes differences in LPL more difficult to detect.

In conclusion, orlistat had no apparent effect on systemic fasting and postprandial HTGL and LPL activities, even after the administration of intravenous heparin. There were also no treatment-related alterations in lipoprotein metabolism detected from the ingestion of orlistat over a 10-d period. The results of this study indicate that the inhibitory effect of orlistat (120 mg t.i.d.) on lipase is limited to the gastrointesinal tract and is not manifested systemically.

1 Blum-Kaelin D, Hill H. Effect of tetrahydrolipstatin (RO 180647/002, THL) on rat, dog and human post-heparin plasma lipolytic activity in vitro. Hoffman-LaRoche GCR 1990; B-103: 367,

2 Kamm JJ. Oncogenicity (feeding) study with RO 180647 in the rat. Hoffmann-LaRoche GCR 1996; N-138: 885,

3 Eckel RH, Goldberg IJ, Steiner L, Yost TJ, Paterniti JR Jr. Plasma lipolytic activity. Relationship to postheparin lipolytic activity and evidence for metabolic regulation. Diabetes 1988; 37: 610-615, MEDLINE

4 Van Gaal LF, Broom JI, Enzi G, Toplak H. Efficacy and tolerability of orlistat in the treatment of obesity: A 6-month dose-ranging study. Orlistat dose-ranging study group. Eur J Clin Pharmacol 1999; 54: 125-132,

5 Glaser DS, Yost TJ, Eckel RH. Preheparin lipoprotein lipolytic activities: Relationship to plasm lipoproteins and postheparin lipolytic activities. J Lipid Res 1992; 33: 209-214, MEDLINE

6 Goers JW, Pedersen ME, Kern PA, Ong J, Schotz MC. An enzyme-linked immunoassay for lipoprotein lipase. Anal Biochem 1987; 166: 27-35, MEDLINE

7 Van Gent CM, van der Voort HA, de Bruyn AM, Klein F. Cholesterol determinations. A comparative study of methods with special reference to enzymatic procedures. Clin Chim Acta 1977; 75: 243-251, MEDLINE

8 Wu LL, Warnick GR, Wu JT, Williams RR, Lalouel J. A rapid micro-scale procedure for determination of the total lipid profile. Clin Chem 1989; 35: 1486-1491, MEDLINE

9 Kohlmeier M. Direct enzymic measurement of glycerides in serum and in lipoprotein fractions. Clin Chem 1986; 32: 63-66, MEDLINE

10 Demacker PNM, Hijmans AGM, Jansen AP. Enzymatic extraction determinations of free fatty acids in serum compared. Clin Chem 1982; 28: 1765-1768, MEDLINE

11 Fernandez M, Borgstrom B. Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat. Biochim Biophys Acta 1989; 1001: 249-255, MEDLINE

12 Zhi J, Melia AT, Eggers H, Joly R, Patel IH. Review of limited systemic absorption of orlistat, a lipase inhibitor, in healthy human volunteers. J Clin Pharmacol 1995; 35: 1103-1108, MEDLINE

13 Drent ML, Larsson I, William-Olsson T, Quaade F, Czubayko F, von Bergmann K, Strobel W, Sjostrom L, van der Veen EA. Orlistat (RO 19-0647), a lipase inhibitor, in the treatment of human obesity: A multiple dose study. Int J Obes 1995; 19: 221-226,

14 Reitsma JB, Castro Cabezas M, de Bruin TW, Erkelens DW. Relationship between improved postprandial lipemia and low-density lipoprotein metabolism during treatment with tetrahydrolipstatin, a pancreatic lipase inhibitor. Metabolism 1994; 43: 293-298. MEDLINE

Figure 1 Hourly levels (mean±s.e.m.) of (A) hepatic triacylglycerol lipase (HTGL), (B) lipoprotein lipase (LPL), and (C) LPL specific activity in plasma after ingestion of a fat-rich breakfast in two groups of normal-weight male subjects, one group given a placebo capsule with breakfast, the other given orlistat 120 mg orally with breakfast. There was no statistical difference between the two groups when (A) postrandial HTGL activity, (B) postprandial LPC acting, and (C) LPL specific activity over 12 h were calculated as total area under the effect curve (AUEC).

Figure 2 Hourly levels (mean±s.e.m.) of serum triglycerides (A), free fatty acids (FFA); (B), HDL-cholesterol (C), LDL-cholesterol (D), and VLDL-cholesterol (E), measured after ingestion of a fat-rich breakfast in two groups of normal-weight male subjects, one group given a placebo capsule with breakfast, the other given orlistat 120 mg orally with breakfast. There was no statistical difference between the two groups in any of the postprandial lipids and lipoproteins when calculated as total area under the effect curve (AUEC).

Table 1 Days 1, 5 and 10: plasma enzymatic and lipid values measured in the fasted state

Table 2 Day 5: areas under the effect curve (AUEC) over the 12 h postprandial period, for preheparin plasma enzymes and plasma lipoproteins

Table 3 Day 10: postheparin lipase values