A high-protein diet increases postprandial but not fasting plasma total homocysteine concentrations: a dietary controlled, crossover trial in healthy voluntee

¹ From the Nutrition and Health Programme, Wageningen Centre for Food Sciences, Wageningen, Netherlands (PV, MRO, and MBK); the Division of Human Nutrition, Wageningen University, Wageningen, Netherlands (PV, MBK, and MRO); and the Business Unit Physiological Sciences, TNO Quality of Life, Zeist, Netherlands (TvV)

² Supported by the Wageningen Centre for Food Sciences, an alliance of major Dutch food industries and research institutions (TNO Quality of Life, Wageningen University and Research Centre, and Maastricht University) that receives financial support from the Dutch government.

³ Reprints not available. Address correspondence to P Verhoef, Division of Human Nutrition, Bomenweg 2, 6703 HD Wageningen, Netherlands. E-mail: petra.verhoef{at}wur.nl.

ABSTRACT  
Background: A high plasma concentration of total homocysteine (tHcy) is associated with increased risk of cardiovascular disease. A high protein intake and hence a high intake of methionine—the sole dietary precursor of homocysteine—may raise plasma tHcy concentrations.

Objectives: We studied whether high intake of protein increases plasma concentrations of tHcy in the fasting state and throughout the day.

Design: We conducted a randomized, dietary controlled, crossover trial in 20 healthy men aged 18–44 y. For 8 d, men consumed a controlled low-protein diet enriched with either a protein supplement [high-protein diet (21% of energy as protein)] or an isocaloric amount of short-chain glucose polymers [low-protein diet (9% of energy as protein)]. After a 13-d washout period, treatments were reversed. On days 1 and 8 of each treatment period, blood was sampled before breakfast (fasting) and throughout the day.

Results: Fasting tHcy concentrations did not differ significantly after the 1-wk high-protein and the 1-wk low-protein diets. The high-protein diet resulted in a significantly higher area under the 24-h homocysteine-by-time curves compared with the low-protein diet, both on day 1 (difference: 45.1 h · µmol/L; 95% CI: 35.3, 54.8 h · µmol/L; P < 0.0001) and on day 8 (difference: 24.7 h · µmol/L; 95% CI: 15.0, 34.5 h · µmol/L; P < 0.0001).

Conclusions: A high-protein diet increases tHcy concentrations throughout the day but does not increase fasting tHcy concentrations. As previously shown, the extent of the tHcy increase is modified by the amino acid composition of the protein diet. The clinical relevance of this finding depends on whether high concentrations of tHcy—particularly postprandially—cause cardiovascular disease.

Key Words: Dietary protein • homocysteine • fasting • postprandial • crossover study • humans

INTRODUCTION  
A high plasma total homocysteine (tHcy) concentration may lead to cardiovascular disease (CVD) (1, 2), but proof that lowering tHcy will prevent these diseases is currently lacking (3). Methionine is the sole precursor of homocysteine, and dietary protein is the main source of methionine. The daily intake of methionine in the general population varies from 1 to 4 g/d (4-6). Intracellularly, methionine is formed into S-adenosylmethionine, which supplies the methyl group for numerous methylation reactions that yield S-adenosylhomocysteine as a product. S-adenosylhomocysteine is metabolized to homocysteine, which is either remethylated to methionine or broken down to cysteine and sulfate in the transsulfuration pathway (7). A single oral dose of free methionine increases the plasma concentration of tHcy in a dose-dependent manner within hours (8). We previously showed that protein-bound methionine (ie, that from a protein-rich meal) increases postprandial plasma concentrations of tHcy but does so more modestly than does the same dose of free methionine (9).

As yet, it is unclear whether habitual consumption of high amounts of protein—and thus of methionine—causes elevated tHcy concentrations. Cross-sectional studies linking habitual protein intakes to fasting tHcy concentrations show conflicting results. Among participants in the Atherosclerosis Risk in Communities study, there was no association between protein or methionine intakes and fasting plasma concentrations of tHcy (10). In another study (11), high habitual protein intakes were associated with lower fasting plasma tHcy concentrations, but those researchers did not adjust for the intakes of vitamins B-6 and B-12, which occur in protein-rich foods and are known to lower plasma tHcy concentrations. In the Framingham Offspring Cohort (12), an initial inverse relation between protein intakes and fasting tHcy concentrations disappeared after adjustment for B vitamin intakes.

Intervention studies in humans have tested effects of high-protein diets or pure methionine supplements. Supplementation of the diet with pure methionine at doses 4–6 times the habitual protein-bound methionine intake (13) appeared to increase fasting tHcy concentrations, whereas lower doses did not (13, 14). The intervention studies comparing high-protein and low-protein intakes showed either no effect (6) or a reduction in fasting tHcy (15). The fact that higher dietary protein intakes did not increase plasma concentrations of fasting tHcy in these studies can be interpreted in several ways. First, like rats (16), humans may adapt to increased methionine intakes by improved homocysteine catabolism, and hence tHcy is completely removed overnight. Second, a protein composition with a high ratio of serine to methionine or of cysteine to methionine may temper the postprandial tHcy rise and hence leave fasting tHcy unaffected (9). Third, the extra intake of B vitamins associated with high protein intakes may counterbalance the tHcy-raising effect of methionine. Therefore, we investigated whether high habitual intake of protein affects the fasting tHcy concentration when the intake of B vitamins and other nutrients is kept constant by consumption of supplements of protein powder instead of consumption of protein-rich foods. We also investigated whether high intakes of protein affect postprandial tHcy concentrations. Like high fasting tHcy concentrations, high tHcy responses after methionine intake are a risk factor for CVD and impaired reactivity of the vascular endothelium (8, 17, 18).

SUBJECTS AND METHODS  
Subjects
Twenty men aged 18–44 y ( All volunteers gave written informed consent. The study was conducted according to Good Clinical Practice guidelines at the Business unit Physiologic Sciences, TNO Quality of Life (Zeist, Netherlands). The protocol was approved by the local medical ethics committee.

Design
The study was designed as a randomized, open, dietary controlled, crossover study in free-living volunteers. Each man underwent 2 dietary treatments: a high-protein diet and a low-protein diet. Each treatment period lasted 8 d, and the intervening washout period, during which the men consumed their habitual diet, lasted 13 d. After the washout period, the treatments were reversed. Half of the men started with the low-protein diet and half with the high-protein diet.

On days 1 and 8 of each treatment period, volunteers stayed in the metabolic ward from early in the morning until 12 h later. On those days, a fasting blood sample was taken just before breakfast (t = 0), and samples were also taken at 2, 4, 6, 8, 10, and 12 h after breakfast. Lunch and dinner were eaten 30 min before the blood samplings at 4 and 10 h, respectively. The next morning (days 2 and 9 of each treatment period), volunteers returned to the institute to provide another fasting blood sample at t = 24 h. On all other treatment days, volunteers consumed only the hot lunch at the research institute and received drinks and foods for a period of 24 h.

The daily diet consisted of the following meals and snacks: a breakfast of bread, diet margarine, jam, ham, colored sprinkles, and a yogurt drink; a morning snack consisting of a glass of cocoa and a biscuit; a lunch consisting of soup, a hot meal (either macaroni with ham and cheese, potatoes with chicken cordon bleu and cauliflower and broccoli, or potatoes with turkey fillet and apples and raisins), custard, and a soft drink; an afternoon snack consisting of a piece of fruit, a candy bar, and another soft drink; a dinner consisting of soup, bread, diet margarine, low-protein toppings (eg, jam and colored sprinkles), orange juice, and custard; and an evening snack consisting of potato chips and a soft drink. The supplements of protein and dextrin-maltose (see Composition of the diet, below) were added to the yogurt drink, cocoa, soup, hot meal, and custard. The protein supplement clearly thickened these drinks and foods, but the dextrine-maltose did not. Consequently, the study was not blinded, as the men had tried a protein-enriched product during the screening visit. The men were not allowed to eat or drink additional food except tap water, coffee, and tea. On the study days in the ward, consumption of coffee and tea was limited to 1 cup (ie, 150 mL) every 2 h and was kept the same on all 4 in-ward study days.

Compliance was measured in several ways. The men registered their consumption of provided foods in a structured diary on a daily basis, recording if, when, and how much they consumed of each item. They were asked to return remains (if any) of the drinks and foods containing the supplements, and the weight of these was registered at the institute. On the 4 whole-day visits to the institute (ie, days 1 and 8 of each treatment period), the personnel checked and registered compliance.

Composition of the diet
Usual energy requirement was estimated from the basal metabolic rate (BMR) and an individual physical activity index (PAI) by the equation

RESULTS  
Men were selected to have normal values for body mass index; plasma tHcy (defined as 20 µmol/L); serum total and HDL cholesterol, triacylglycerol, and creatinine; and blood pressure (Table 2). At screening, there was no difference in mean values of these variables or in mean fasting tHcy concentrations between the 10 men who started with the high-protein treatment and the 10 men who started with the low-protein treatment (data not shown). Reported and observed compliance and adherence to the dietary restrictions were generally very good. The men were reported to have consumed 99.6% of the protein supplement and 99.8% of the dextrin-maltose supplement.

View this table:
TABLE 2. Characteristics at screening of all 20 men included in the study¹

 
Mean fasting plasma tHcy concentrations on day 8 did not differ significantly between the diets: they were 9.3 ± 2.1 µmol/L after the high-protein treatment and 9.2 ± 2.2 µmol/L after the low-protein treatment (difference: 0.1 µmol/L; 95% CI: –0.3, 0.5 µmol/L; P = 0.5). The treatments showed very distinct effects on the plasma tHcy concentrations measured over a 24-h period (Figure 1). During the high-protein diet, tHcy concentrations rose steadily after breakfast and lunch, reached a peak before dinner, and returned to normal the next morning. Patterns were not materially different on days 1 and 8. During the low-protein diet, plasma tHcy concentrations throughout the day either were lower than in the fasting state on day 1 or showed a very modest increase after breakfast, which was followed by decreases after lunch and dinner (on day 8). On both days, tHcy concentrations returned to normal overnight. Consequenly, the high-protein diet resulted in a significantly higher 24-h AUC than did the low-protein diet on both days 1 and 8 (Table 3). Furthermore, the maximum change was significantly higher after the high-protein diet than after the low-protein diet on both days 1 and 8 (Table 3).

View larger version (17K):
FIGURE 1.. Mean plasma total homocysteine concentrations among 20 men in the fasting state (t = 0 h) and at various time points (t = 2, 4, 6, 8, 10, 12, and 24 h), during either a high-protein treatment (, ) or a low-protein treatment (, •) on day 1 (, •) and day 8 (, ) of the treatment periods.

 

View this table:
TABLE 3. Maximum change in plasma total homocysteine concentration from baseline (_max) and the 24-h area under the homocysteine-by-time curve (AUC) during high-protein and low-protein diets on day 1 and day 8 of the treatment periods

 
Mean fasting triacylglycerol concentrations were significantly higher (P = 0.001, based on ln-transformed data) after the 1-wk low-protein diet (1.8 ± 0.7 mmol/L; range: 0.7–3.7 mmol/L) than after the 1-wk high-protein diet (1.4 ± 0.7 mmol/L; range: 0.5–4.0 mmol/L). Mean fasting concentrations of other blood lipids and mean blood pressure in the fasting state did not differ significantly between the treatments, and fasting blood concentrations of vitamins B-6 and B-12 and folate did not change during the study. Creatinine concentrations and estimated creatinine clearances of all subjects were within the normal ranges. Over the entire study period of 29 d, weight increased by 1.2 ± 1.1 kg (P = 0.0002, paired t test).

DISCUSSION  
In this crossover study among 20 healthy men, we found that a diet containing 21% of energy as protein, or 4–4.5 g protein-bound methionine/d, increased plasma tHcy concentrations throughout the day, both acutely and after 1 wk of habituation. Fasting plasma concentrations of tHcy were not affected by the 1-wk high-protein diet, which indicates that concentrations had returned to baseline after an overnight fast. This is consistent with the half-life of 4 h that has been reported for homocysteine in humans (26). During the 1-wk low-protein diet (9% of energy from protein, or 1.7 g protein-bound methionine/d), plasma tHcy concentrations throughout day 1 were lower than those in the fasting state, and, on day 8, they showed a very modest increase after breakfast and decreases after lunch and dinner.

Unlike investigators in previous studies, we were able to separate the effect of a high intake of protein from the increased intake of B vitamins that is usually associated with high protein intakes. In addition, we were the first to study the effect of a high protein intake on plasma concentrations of tHcy measured throughout the day. The importance of high postprandial tHcy concentrations as a possible cause of CVD is indicated by other studies. First, in epidemiologic studies, the tHcy concentrations after a classical postmethionine load, although greater than those after dietary protein-bound methionine, were associated with CVD independently from the fasting concentrations (17). Second, it was observed that free methionine or methionine from a meal acutely impaired the reactivity of the vascular endothelium (8, 18), which is a promising early marker of CVD risk.

Our finding that fasting tHcy was not influenced by protein intake confirmed the results of 2 observational studies (10, 12). Stolzenberg-Solomon et al (11) reported an inverse association between protein intake and fasting tHcy, but this difference from our findings may be explained by the fact that those investigators failed to adjust for the higher intakes of vitamins B-6 and B-12 that are associated with high protein intake. Only 2 other (dietary) intervention studies have tested the effect of increased protein intakes on fasting plasma concentrations of tHcy (6, 15). Ward et al (6) shifted the subjects with low habitual protein intake to high protein intake and vice versa. The maximum change in protein-bound methionine intake that was obtained in their study was 1.5 g/d, which was only about one-half the change in our study. Like us, Ward et al (6) found no effect of changes in protein intake on fasting concentrations of plasma tHcy. Haulrik et al (15) studied the effect of changes in protein intake on body weight and fasting concentrations of plasma tHcy. Compared with the intermediate-protein diet (14% of energy as protein), the high-protein diet (22% of energy as protein) lowered fasting homocysteine by 25%, whereas the low-protein diet (12% of energy as protein) had no effect. However, interpretation of these findings is hampered by the fact that the subjects in the high-protein group started out with higher tHcy concentrations and lost more weight than did those in the low-protein group (9.4 and 5.9 kg, respectively). It is not clear whether weight reduction affects tHcy concentration. Experimental data, which are limited to obese subjects undergoing gastric restrictive surgery, show that weight reduction actually increases tHcy in that particular group of patients (27, 28), which made it unlikely that weight loss was the reason for the drop in tHcy concentrations in the high-protein group studied by Haulrik et al (15). However, neither they nor Ward et al (6) could separate the effect of increased protein intake from that of increased intake of B vitamins. In our study, the intake and blood concentrations of B vitamins were constant throughout the study period.

Two research groups fed the subjects supplemental methionine as a proxy for high protein intake (13, 14). Unfortunately, neither of those studies included a group of control subjects. Andersson et al (14) observed no effect on fasting or postload tHcy concentrations of a 2-wk supplementation with methionine at breakfast (300% of normal or 3 g instead of 1 g/d). Ward et al (13) supplemented methionine at doses of 2, 4, and 6 times the habitual intake in a sequential design. The fasting tHcy concentration was unaffected by the lowest dose, but increased by 60% and 100% at the 2 higher doses of methionine, respectively. Apparently these high-dose methionine supplements induced increases in tHcy concentrations that persisted until the next morning, unlike the high-protein meals used in our study. The other amino acids present in the protein-rich meal, eg, cysteine and serine, may explain part of this difference (9).

Several aspects of our study design require further discussion. First, there was an unintended mean weight gain of 1.2 kg over the entire study period, which occurred because the dietitian did not take into account the amount of energy provided by the study substances. Hence, the men received a diet with an energy content 0.4–1.5 MJ above their daily estimated energy requirement. However, the fact that we applied a randomized crossover design made it unlikely that this weight gain would explain the observed effects on the plasma concentrations of tHcy.

Second, we chose a high-carbohydrate diet for control treatment because a high-fat diet seemed unfeasible and unacceptable. There was hardly any increase in tHcy concentrations after consumption of the high-carbohydrate meals. We assume that this observation was explained by the low protein content of the high-carbohydrate diet. Insulin effects seem less likely because increases in insulin evoked by raised serum glucose reduce expression of cystathionine ß-synthase and hence reduce homocysteine breakdown (29). An explanation for the curve of the low-protein diet being lower on day 1 than on day 8 may be that habitual protein intake before the start of the study—ie, 15% (according to the data the 3rd Dutch National Food Consumption Survey, conducted in 1997–1998)—was greater than the intake of 9% of energy on the low-protein diet. It is likely that these men had a higher rate of homocysteine breakdown through the transsulfuration at the beginning of the low-protein period than at the end.

Third, replacement of dietary proteins by carbohydrates increased the serum concentration of triacylglycerols, which is in line with the findings of a previous study (30). Replacement of dietary fat by carbohydrates also increases triacylglycerols (31). This observation indicates, indirectly, that participants complied with the diet; however, compliance was verified by daily registration of consumption of provided supplements and by checking the returned remains. One week was probably too short a time in which to see the decrease in serum HDL cholesterol that is usually observed during a high-carbohydrate diet, because HDL cholesterol is known to respond more slowly than do triacylglycerols (32).

The current US dietary reference intakes established by the Institute of Medicine (33) allow that 10–35% of total energy is provided by protein. On the basis of our present findings, the upper limit of 35% could be considered too high. Of course, the public health relevance of our observation depends on whether high plasma tHcy is a cause of CVD and other chronic diseases. Two secondary prevention trials showed no reduction in coronary heart disease or stroke after 2 y of tHcy-lowering treatment (34, 35), but, in a few years, data will be available from 50 000 patients who were allocated to receive B vitamins or placebo (36).

In conclusion, compared with a high-carbohydrate diet, a high-protein diet raises tHcy concentrations throughout the day, both acutely and after 1 wk of habituation. Fasting tHcy concentrations are not affected by a high-protein diet. As we previously showed, the extent of the postprandial rise in tHcy is likely to be modified by the amino acid composition of the protein diet. The clinical relevance of our finding depends on whether high concentrations of tHcy—in particular, high postprandial concentrations—do indeed cause CVD. If so, it might be important to avoid excessive protein intake.

ACKNOWLEDGMENTS  
We thank the volunteers for their participation, all those involved in the conduct of the experiment at Business Unit Physiologic Sciences, TNO Quality of Life (Zeist, Netherlands), for their dedication, and the laboratory staff at Wageningen University and TNO Quality of Life for careful analyses. Furthermore, we thank the Department of Chronic Disease Epidemiology, National Institute of Public Health and the Environment, Bilthoven, Netherlands, for data on the amino acid composition of foods.

All authors were responsible for the design of the study. TvV was in charge of the conduct of the experiment, including selection of subjects, planning, and implementation. PV prepared several drafts of the manuscript. All other authors critically revised the manuscript. None of the authors had a financial or personal conflict of interest.

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