Hormonal response to diets high in soy or animal protein without and with isoflavones in moderately hypercholesterolemic subjects

Consumption of soy protein has been associated with altered risk of developing endocrine-regulated cancers. This study was designed to assess the independent effect of soy relative to animal protein and soy-derived isoflavones on circulating estrogen and androgen concentrations in postmenopausal women and older men. Forty-two subjects (> 50 yr) with low-density lipoprotein cholesterol levels of > or = 3.36 mmol/l were fed each of 4 diets in randomized order for 6 wk/phase. All food and drink were provided. Diets contained 25 g soy or common sources of animal protein/4.2 MJ containing trace or 50 mg isoflavones/4.2 MJ. At the end of each diet phase, concentrations of estrone sulfate, estrone, estradiol, testosterone, androstendione, dihydrotestosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate were measured. In postmenopausal women, concentrations of estrone were higher and its precursor, dehydroepiandrosterone, lower after consuming the soy compared with animal protein diets (P = 0.0396 and 0.0374, respectively). There was no significant effect of isoflavones on any of the hormones measured. In older men, dehydroepiandrosterone sulfate concentrations were lower after consuming the isoflavone (P = 0.0106) and higher after soy, compared with the animal protein diets (P = 0.0118). These data suggest that relatively large amounts of soy protein or soy-derived isoflavones had modest and limited sex-specific effects on circulating hormone levels.     

The effect of soy protein with or without isoflavones relative to milk protein on plasma lipids in hypercholesterolemic postmenopausal women

BACKGROUND: Clinical trial data and the results of a meta-analysis suggest a hypocholesterolemic effect of soy protein. The effect may be partially attributable to the isoflavones in soy. Few studies have examined the separate effects of soy protein and isoflavones. OBJECTIVE: The objective of this study was to determine the effect of soy protein and isoflavones on plasma lipid concentrations in postmenopausal, moderately hypercholesterolemic women. DESIGN: This was a randomized, double-blind, placebo-controlled clinical trial with 3 treatment groups. After a 4-wk run-in phase during which the women consumed a milk protein supplement, the subjects were randomly assigned to 12 wk of dietary protein supplementation (42 g/d) with either a milk protein (Milk group) or 1 of 2 soy proteins containing either trace amounts of isoflavones (Soy- group) or 80 mg aglycone isoflavones (Soy+ group). RESULTS: LDL-cholesterol concentrations decreased more in the Soy+ group (n = 31) than in the Soy- group (n = 33) (0.38 compared with 0.09 mmol/L; P = 0.005), but neither of these changes was significantly different from the 0.26-mmol/L decrease observed in the Milk group (n = 30). The results for total cholesterol were similar to those for LDL cholesterol. There were no significant differences in HDL-cholesterol or triacylglycerol concentrations between the 3 groups. CONCLUSIONS: The difference in total- and LDL-cholesterol lowering between the 2 soy-protein supplements suggests an effect attributable to the isoflavone-containing fraction. However, the unexpected LDL-cholesterol lowering observed in the Milk group, and the fact that there was no significant difference between either soy group and the Milk group, suggests that changes may have been due to other factors related to participation in the study.     

Snack bars with high soy protein and isoflavone content for use in diets to control dyslipidaemia

The objective was to develop a cereal bar product rich in isoflavones and soy protein to be used in diets for controlling dyslipidaemia. A soy snack bar with 39.88 g/100 g dietary fibre, 34.25 g/100 g protein, 100.39 mg/100 g isoflavones and 245.47 kcal/100 g was produced. The shelf life of the product was tested over a period of 6 months. The hardness, water activity and darkness of the snack bars increased with storage time. Clinical trial was carried out to evaluate the effectiveness of the soy snack bar on the lipid profiles of 22 dyslipidaemic subjects for a period of 45 days. There was a decrease in levels of triglycerides (TGs) (- 20%) and an increase in high-density lipoprotein cholesterol (HDL-c) (+8%) comparing with baseline values. Therefore, relatively high intakes of soy protein and isoflavones in moderately hypercholesterolaemic subjects (>200 mg/dl) may have a moderately beneficial effect on plasma lipid profiles, as TG and HDL-c levels, without additional diet restrictions.     

Lipid response to a low-fat diet with or without soy is modified by C-reactive protein status in moderately hypercholesterolemic adults

Recent evidence suggests that individuals with high concentrations of C-reactive protein (CRP), a marker of inflammation, are less responsive to cholesterol-lowering diets. CRP concentrations are increased by oral estrogen; however, the effect of soy phytoestrogens on inflammation has not been studied comprehensively, especially in women receiving hormone replacement therapy (HRT). This study was conducted to determine whether adding soy to a low-fat, high-fiber diet affects CRP and interleukin (IL)-6, and to examine the association between CRP levels and lipid response in moderately hypercholesterolemic adults (men = 18, postmenopausal women = 14; 6 receiving HRT). After a 3-wk run-in period with consumption of a Step I diet (27% total fat, 7% saturated fat, 275 mg cholesterol), participants were randomly assigned to diets containing 25 g/d soy protein (+ 90 mg/d isoflavones) or 25 g/d milk protein for 6 wk in a crossover design. Lipids and lipoproteins, CRP, and IL-6 were measured at the end of each diet and participants were categorized into high (>3.5 mg/L) or low CRP groups based on a median split. The addition of soy or milk protein to the Step I diet did not affect lipids or inflammatory markers. Regardless of protein source, those with low CRP exhibited significant decreases in LDL cholesterol (-3.5%) and the LDL:HDL cholesterol ratio (-4.8%), whereas those with high CRP had significant increases in LDL cholesterol (+4.8%), the LDL:HDL cholesterol ratio (+5.2%), apolipoprotein B (+3.8%), and lipoprotein(a) (+13.5%) compared with the run-in diet. These results suggest that inflammation may not only attenuate lipid responses, but also aggravate dyslipidemia in hypercholesterolemic subjects consuming a cholesterol-lowering diet.     

Effects of feeding rats low protein diets containing casein or soy protein isolate supplemented with methionine or oligo-L-methionine

The effects of supplementing 8% casein or 10% soy protein isolate (SPI) diets with graded levels of oligo-L-methionine (a mixture of hexa- and heptapeptides, OM) or L-methionine (Met) were studied in rats to determine the reason for the difference in nutritional quality between proteins and corresponding amino acid mixtures. As the OM concentration of the casein-based diet was increased from 0.02% to 0.6%, maximum weight gain was attained at 0.2%, and the growth-promoting activity of OM was comparable to Met at all the corresponding levels tested. Liver fat began to accumulate when supplemental Met reached a level of 0.08% of the casein diet, but OM addition did not produce a fatty liver at dietary levels of less than 0.3%. When SPI was used as the dietary protein source, the effect of supplemental OM was significantly less than that of Met. Digestibility of OM (assessed by incremental portal plasma Met concentration) was measured 30 min after feeding the casein or SPI diet supplemented with 3% OM using rats fasted for 24 h. Plasma Met concentration was greatly increased in rats fed the casein plus OM diet compared with that of rats fed the SPI + OM diet. Similarly, the 30-min portal Met concentration significantly increased in response to the casein + OM diet compared with the SPI + OM diet regardless of the prefed proteins (25% casein and 25% SPI for 2 wk).(ABSTRACT TRUNCATED AT 250 WORDS)     

Health effects of soy protein and isoflavones in humans

Epidemiological investigations suggest that soy consumption may be associated with a lower incidence of certain chronic diseases. Clinical studies also show that ingestion of soy proteins reduces the risk factors for cardiovascular disease. This led to the approval of the food-labeling health claim for soy proteins in the prevention of coronary heart disease by the U.S. FDA in 1999. Similar health petitions for soy proteins have also been approved thereafter in the United Kingdom, Brazil, South Africa, the Philippines, Indonesia, Korea, and Malaysia. However, the purported health benefits are quite variable in different studies. The Nutrition Committee of the American Heart Association has assessed 22 randomized trials conducted since 1999 and found that isolated soy protein with isoflavones (ISF) slightly decreased LDL cholesterol but had no effect on HDL cholesterol, triglycerides, lipoprotein(a), or blood pressure. The other effects of soy consumption were not evident. Although the contributing factors to these discrepancies are not fully understood, the source of soybeans and processing procedures of the protein or ISF are believed to be important because of their effects on the content and intactness of certain bioactive protein subunits. Some studies have documented potential safety concerns on increased consumption of soy products. Impacts of soy products on thyroid and reproductive functions as well as on certain types of carcinogenesis require further study in this context. Overall, existing data are inconsistent or inadequate in supporting most of the suggested health benefits of consuming soy protein or ISF.     

Iron indexes and total antioxidant status in response to soy protein intake in perimenopausal women

BACKGROUND: Elevated iron stores, oxidative stress, and estrogen deficiency may place postmenopausal women at greater risk of heart disease and cancer than premenopausal women. OBJECTIVE: The objective was to determine the effect of soy-protein isolate (SPI) intake and iron indexes on plasma total antioxidant status (TAS) in perimenopausal women after control for other contributing factors. DESIGN: Perimenopausal women (n = 69) were randomly assigned (double blind) to treatment: isoflavone-rich SPI (SPI+; n = 24), isoflavone-poor SPI (SPI-; n = 24), or whey protein (control; n = 21). Each subject consumed 40 g soy or whey protein daily for 24 wk. Plasma TAS, serum ferritin, serum iron, transferrin saturation, and hemoglobin were measured at baseline, week 12, and week 24. RESULTS: No significant time-by-treatment interactions on iron indexes or TAS were observed, whereas time had an effect on serum ferritin (P < or = 0.0001) and hemoglobin (P = 0.004) but not on TAS. Multiple regression analysis showed that at week 12, 48% (P < or = 0.0001) of the variability in TAS was accounted for by baseline TAS, alcohol intake, soy intake (soy compared with control; P = 0.016), plasma lipoprotein(a), and dietary iron. At week 24, 47% of the variability in TAS was accounted for by baseline TAS, serum ferritin, serum estrone, dietary zinc, and dietary meat, fish, and poultry. CONCLUSIONS: SPI intake had no significant effect on iron status, but our results suggest that dietary soy protein and low iron stores may protect perimenopausal women from oxidative stress.     

Changes in plasma antioxidant status following consumption of diets high or low in fruit and vegetables or following dietary supplementation with an antioxidant mixture

The aim of the present study was to examine the effect of consumption of a high-fruit and vegetable diet, or a spray-dried extract of selected fruits and vegetables of high antioxidant content, on indices of antioxidant status of individuals consuming a background diet with minimal antioxidant intake. Plasma antioxidant concentrations were determined in twenty-five men following a 2-week depletion period during which they consumed self-selected low-antioxidant diets (less than three servings of fruit and vegetables with no tea, coffee, red wine or fruit juice). Following this period the volunteers consumed either a self-selected diet containing five to seven servings of fruit and vegetables/d, or 30 g of a spray-dried supplement designed to provide the equivalent antioxidant activity of five to seven servings of fruit and vegetables for 2 weeks in a crossover trial. Following consumption of a high-antioxidant diet for 2 weeks, plasma concentrations of ascorbic acid, alpha- and beta-carotene and lutein+zeaxanthin were all significantly increased (P < 0.05) over the depletion period. However, concentrations of lycopene, retinol and tocopherol were not affected. Consumption of the supplement also raised the concentrations of these same antioxidants in plasma. Despite the increases in the concentrations of measured antioxidant nutrients, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid-equivalent antioxidant capacity of plasma, as estimated by inhibition of metmyoglobin activity, was not significantly affected by any of the dietary treatments.     

Blood lipid and oxidative stress responses to soy protein with isoflavones and phytic acid in postmenopausal women

BACKGROUND: Postmenopausal women are at risk of cardiovascular disease (CVD) as a result of unfavorable blood lipid profiles and increased oxidative stress. Soy protein consumption may help protect against these risk factors. OBJECTIVE: Our objective was to ascertain the effect of the soy protein components isoflavones and phytate on CVD risk in postmenopausal women. DESIGN: In a double-blind 6-wk study, 55 postmenopausal women were randomly assigned to 1 of 4 treatments with soy protein (40 g/d) isolate (SPI): low phytate/low isoflavone (LP/LI); normal phytate/low isoflavone (NP/LI); low phytate/normal isoflavone (LP/NI); or normal phytate/normal isoflavone (NP/NI). Blood lipids (total, LDL, and HDL cholesterol and triacylglycerol) and oxidative stress indexes (protein carbonyls, oxidized LDLs, and 8-iso-prostaglandin-F(2alpha)) were measured at baseline and 6 wk. RESULTS: The oxidative stress indexes were not significantly affected by either phytate or isoflavones. Phytate treatment had a minimal but nonsignificant effect in reducing protein carbonyls and 8-iso-prostaglandin-F(2alpha); the reductions were 6-8% and 4-6% in the NP/LI and NP/NI groups and 1-4% and 3-4% in the LP/LI and LP/NI groups, respectively. Similarly, circulating lipids were not significantly affected by either phytate or isoflavones. The decline in total (6%-7% compared with 2%-4%) and LDL (10%-11% compared with 3%-7%) cholesterol did not differ significantly between the normal- and low-isoflavone groups, respectively. CONCLUSION: In postmenopausal women, neither phytate nor isoflavones in SPI have a significant effect of reducing oxidative damage or favorably altering blood lipids.     

Plasma amino acids and excretion of protein end products by mice fed 10 or 40% soybean protein diets with or without dietary benzo[a]pyrene or 1,2-dimethylhydrazine

The effects of two carcinogens benzo[a]pyrene (BP) and symmetrical 1,2-dimethylhydrazine dihydrochloride (DMH), on plasma amino acid concentrations and on excretion of lipids and nitrogenous metabolites were studied in 7- to 8-wk-old male and female B6C3F1 mice. BP and DMH were fed at concentrations of 0.3125 and 0.0225 g/kg, respectively, in purified diets containing 10 or 40% soybean protein. Nutritional balances were measured over a 7-d period after 7 d of acclimatization. Females excreted less urea and more NH3 than males. Urinary urea-nitrogen, NH3, allantoin, uric acid and total urinary nitrogen were consistently higher in mice fed 40% protein than in those fed 10% protein. The increases in total and NH3 nitrogen paralleled the increase in nitrogen intake. Nitrogen of urea rose more, while that of allantoin and uric acid rose less, than nitrogen intake. Fecal lipid excretion, as a percentage of intake, was consistently higher in mice fed the 40% protein diets than in mice fed 10% protein. Plasma glycine and branched-chain amino acids were higher, but citrulline was lower, when the 40% protein diet was fed. Body weight gain was higher when the 10% protein diet was fed with BP than without it, but BP made no apparent difference in weight gain when the 40% protein diet was fed. BP interacted with dietary protein to influence the excretion of nitrogenous metabolites. In addition, BP feeding produced numerous BP X sex and BP X protein interactions for plasma amino acid concentrations. Compared to controls, feed intake and weight gain were, respectively, 8 and 61% lower in DMH-fed animals during wk 1, but no differences in intake or weight gain were found during wk 2. In contrast to BP, DMH had no significant effects on urinary or fecal nitrogen metabolites, except that urinary uric acid (relative to nitrogen intake) was 9% higher in DMH-fed mice than in controls. DMH-fed mice had 43% higher serum glutamate and 6% lower glutamine than controls.     

Antioxidant action of soy isoflavones on oxidative stress and antioxidant enzyme activities in exercised rats

BACKGROUND/OBJECTIVES

Isoflavones are widely believed to be beneficial to human health, in relation to their antioxidant potentials. Exercise can cause an imbalance between reactive oxygen species (ROS) and antioxidants. This study was conducted in order to investigate the ability of isoflavones in amelioration of oxidative stress induced by exercise.

MATERIALS/METHODS

Male Sprague-Dawley rats were assigned to one of four groups: isoflavone-free with no exercise (CON-sd), isoflavone-free with exercise (CON-ex), isoflavone-supplemented with no exercise (ISF-sd), and isoflavone-supplemented with exercise (ISF-ex). Animals exercised on the treadmill for 30 minutes per day, five days per week. TBARS as a marker of oxidative stress and antioxidant enzyme activity, including SOD, GSH-px, and catalase were determined in liver tissue. Serum lipid profile was also examined.

RESULTS

A significant effect of isoflavone alone was observed on abdominal fat pad mass. ISF-ex had significantly less abdominal fat pad than CON-ex. Both exercise and isoflavone treatment had significant effects on lowering plasma triglyceride (TG), thus, the ISF-ex group had a significantly lower TG level than the CON-sd group, by 30.9%. However, no differences were observed in plasma cholesterol, HDL-C, and cholesterol/HDL-C ratio. Exercise, isoflavone, and exercise-isoflavone interaction effects were significant on thiobarbituric acid reactive substances (TBARS) (P = 0.001, 0.002, and 0.005, respectively). The CON-ex group showed a higher TBARS level than the other three groups. By contrast, in the ISF-ex group, TBARS was restored to the level of the ISF-sd or CON-sd group. Isoflavone had a significant effect on superoxide dismutase (SOD) (P = 0.022) and catalase activities (P = 0.049). Significantly higher SOD and catalase activities were observed in ISF-ex than CON-ex. SOD and catalase activities showed an inverse pattern of TBARS. Taken together, isoflavones increased the activities of SOD and catalase with concomitant decreases in TBARS, indicative of decreased oxidative stress.

CONCLUSIONS

Isoflavone supplementation enhances antioxidant action with attenuation of exercise-induced oxidative stress, as measured by decreases in TBARS, and inhibits body fat accumulation and plasma TG increase. Antioxidative effects ascribed to isoflavones may be partially exerted via enhancement of antioxidant enzyme activities.     

Chemopreventive effects of soy protein and purified soy isoflavones on DMBA-induced mammary tumors in female Sprague-Dawley rats

There are conflicting reports on the effect of soy and its components on mammary carcinogenesis in adult female rats, mainly because of different rodent models that are used in chemoprevention studies. The present study was undertaken to compare the tumor-preventative effects of soy protein isolate (SPI) and two of its isoflavones in a "standard" model that had been used for the identification of many chemopreventive agents. Six groups of female Sprague-Dawley rats were provided with modified cornstarch AIN-76A diets supplemented as follows: no additional agents (control), purified genistein (200 mg/kg diet), purified daidzein (200 mg/kg diet), genistein + daidzein (100 mg/kg diet each), SPI containing normal levels of isoflavones (SPI-n), or SPI depleted of isoflavones (SPI-d). Mammary carcinomas were induced by 7,12-dimethylbenz[a]anthracene (DMBA) introduced 1 wk after the animals began consuming the experimental diets. At the end of the study (120 days after DMBA treatment), no significant differences were found among the six groups with respect to tumor incidence or survival, nor was there a significant reduction in tumor multiplicity in the genistein or genistein + daidzein group. However, there was a 32% reduction in tumor multiplicity in the daidzein and SPI-n groups relative to the control group (P < 0.05). The most effective diet was SPI-d, which produced a 50% reduction in tumor multiplicity relative to the control (P < 0.01). The difference between the SPI-d group and the daidzein or SPI-n group was not significant. Median tumor latency was increased from 53 days in the control group to 68 days in the daidzein group and to 72 days in the SPI-d group, but these differences were not statistically significant. These results show that daidzein and SPI (with normal or low levels of isoflavones) are effective inhibitors of DMBA-induced mammary tumors in adult rats.     

Effects of heat treatment on structural modification and in vivo antioxidant capacity of soy protein

ObjectiveThe present study identified the effects of heat oxidation on protein carbonyl content and α,α-diphenyl-β-picrylhydrazyl (DPPH) free radical-scavenging activity in soy protein. The changes on antioxidant status in male mice fed a heat-oxidized diet were also investigated.MethodsSoy protein heated at 100°C for 30, 60, and 90 min was used to determine the protein carbonyl content and DPPH free radical-scavenging activity in vitro. Male KM mice (3 wk old) were fed a normal diet, an oxidized diet (HD) containing 12% heat-oxidized soy protein, or an HD supplemented with 0.1% lipoic acid. After 4 wk of feeding, apparent digestibility, reactive oxygen species, malondialdehyde, and total antioxidant capacity levels were measured. The antioxidant enzyme activities in serum and tissues were also assayed.ResultsHeat-oxidized soy protein showed a significant increase in protein carbonyl formation and a decrease in DPPH free radical-scavenging activity. The HD induced a significant decrease in food intake and apparent digestibility of dry matter and crude protein in mice. Increased levels of reactive oxygen species and malondialdehyde in serum and tissues accompanied by decreased total antioxidant capacity and antioxidant enzyme activities were also observed in HD-fed mice. These changes were partly restored in the lipoic acid–treated group.ConclusionHeat-oxidized soy protein showed a relatively higher protein carbonyl content and a loss of its free radical-scavenging activity in vitro. The heat oxidation also led the soy protein to generate reactive oxygen species, decrease the antioxidant status, and induce redox imbalance in vivo. The heat oxidation of food protein could be a potential health risk in humans.     

Pancreatic response to long-term feeding of soy protein isolate, casein or egg white in rats

Rats were fed purified diets which provided 24% protein from casein (C), soy protein isolate (SPI), or egg white (EW) for 18 mo. Groups of rats were killed at 3, 6, 12 and 18 mo; the pancreata were removed and examined histologically for occurrence of atypical nodules. The weight, protein, DNA, trypsin and chymotrypsin concentrations of the pancreas at each period were measured. Over the entire experimental period, body weight did not differ among groups. Pancreatic weight, protein and trypsin activity were highest in the EW group, followed by the SPI group, and lowest in the C group. Chymotrypsin activity was significantly higher in the EW and SPI groups than the C group. DNA content did not differ significantly among groups over the entire experimental period, although it was elevated in the SPI or EW groups compared to the C groups at some of the time periods. Only one microscopic nodule was observed in all of the animals; it was found at 3 mo in the pancreas from an animal fed EW. Overall, the results suggest that the elevation in enzyme activity and pancreatic weight associated with long-term consumption of EW and SPI did not result in development of pancreatic lesions in rats.     

Dietary soy protein isolate and isoflavones modulate hepatic thyroid hormone receptors in rats

Thyroid hormone receptors (TRs) are regulators of many genes involved in cholesterol and lipid metabolism. The purpose of this study was to examine the effect of soy protein isolate (SPI) and isoflavones on hepatic TRs in rats. In Expt. 1, Sprague-Dawley rats were fed diets containing either casein or alcohol-washed SPI with or without isoflavone supplementation (5-1250 mg/kg diet) for 70, 190, and 310 d. The offspring (F1) were fed the same diets as their parents (F0). In Expt. 2, Sprague-Dawley rats were fed diets containing casein or casein plus isoflavones (50-400 mg/kg diet) for 120 d. The mRNA and protein contents of the hepatic TRs were measured by semiquantitative RT-PCR and Western blot, respectively. TRalpha1, TRalpha2, and TRbeta2 contents were not affected by SPI. However, the content of the 52-kDa TRbeta1 protein, the major isoform present in the liver, was markedly increased by dietary SPI in both sexes of F0 and F1 compared with casein. The supplemental isoflavones had no effect on TRbeta1, whereas the high doses of isoflavones (250 and 1250 mg/kg diet) reduced the hepatic TRalpha1 protein content in F1 male rats on d 28. SPI had no effect on total T3 and T4 levels. However, higher dose of supplemental isoflavones markedly increased T4 level in female rats. Overall, this study demonstrates for the first time that SPI upregulates hepatic TRbeta1 expression, and that isoflavones reduce the hepatic TRalpha1 level in young male rats. The SPI-induced TRbeta1 may play a role in mediating the hypocholesterolemic and lipid-lowering actions of soy protein.     

Plasma amino acids and cholesterol following consumption of dietary casein or soy protein in minipigs

Numerous investigators have claimed that protein-induced differences in plasma cholesterol are mediated by differences in amino acid composition. We have explored whether the venous postprandial amino acid profile reflects differences in the amino acid composition of the protein consumed. Six adult G?ttingen miniature pigs were fed a semisynthetic diet based on either casein or soy protein isolate. Frequent blood sampling was performed over a whole day after consumption of each diet for 6 wk. Postprandial plasma amino acid concentrations reached their maxima within the first 3 h. A group of eight protein amino acids (Met, Arg, Tyr, Val, Trp, Leu, Lys and Cys) exhibited the most marked and significant protein-dependent differences during this early postprandial phase, whereas Thr and His showed less marked differences. With one exception (Ser) all protein amino acids exhibited venous plasma concentration changes in qualitative accordance with their content in the dietary protein consumed. In quantitative terms, however, venous plasma amino acid changes were less marked than expected from the amino acid composition of the dietary proteins. We conclude that neither the considerable number of amino acids showing differences as reported herein nor the multitude of contradictory reported by others concerning single amino acids affecting serum cholesterol favor the hypothesis that one or several amino acid(s) cause protein-induced hypercholesterolemia.