Application of a [13CO2] breath test to study short-term amino acid catabolism during the postprandial phase of a meal

A [13CO2] breath test was applied as a non-invasive method to study the catabolism of ingested amino acids shortly after a meal. This test requires the ingestion of a [1-13C]-labelled amino acid and the analysis of expired air for [13C] enrichment and CO2. The recovery of label as [13CO2] reflects the catabolism of the [1-13C]-labelled substrate. Such a non-steady state approach provides information that is complementary to the information obtained by steady-state methods using a primed continuous infusion of tracer amino acids during the fed state. In a model study with twenty adult male rats, two groups of animals were fed twice a day with one of two semi-synthetic iso-energetic diets. One diet contained egg white protein (EW) as the sole amino acid source. The second diet contained a mixture of free amino acids with a pattern similar to that of the EW diet. On day 5 of the dietary treatment, L-[1-13C]leucine, either bound in EW protein or in free form, was ingested as part of the morning meal. The expired air was sampled at 30 min intervals for 5 h. The rate of recovery ranged from 0% to 6% of the dose/h. Up to 120 min after the onset of the meal, the recovery values for the free amino acid diet were higher than those for the EW diet. Differences in recovery reflect differences in postprandial utilisation. The differences in label recovery were mainly determined by the [13C] enrichment of the expired air. As a consequence, CO2 measurements are not mandatory when CO2 production is comparable.     

Greater stimulation of myofibrillar protein synthesis with ingestion of whey protein isolate v. micellar casein at rest and after resistance exercise in elderly men

We aimed to determine the effect of consuming pure isolated micellar casein or pure whey protein isolate on rates of myofibrillar protein synthesis (MPS) at rest and after resistance exercise in elderly men. Healthy elderly men (72 (sem 1) years; BMI 26·4 (sem 0·7)?kg/m2) were divided into two groups (n 7 each) who received a primed, constant infusion of l-[ring-13C6]phenylalanine to measure MPS at rest and during 4?h of exercise recovery. Participants performed unilateral leg resistance exercise followed by the consumption of isonitrogenous quantities (20?g) of casein or whey. Blood essential amino acids and leucine concentration peaked 60?min post-drink and were greater in amplitude after whey protein ingestion (both, P?相似文献   

The dispersion state of milk fat influences triglyceride metabolism in the rat

Summary Background Milk fat, which has different structures in the various dairy products, is a major and controversial lipid source in the Western diet. However, information about the digestion fate of milk fat depending on its supramolecular structure for a given composition is scarce. Aim of the study In this study, ¹³CO₂ breath tests were performed with fasted rats force–fed different dairy preparations of similar composition but differing in fat suprastructure in order to highlight differences of general lipid metabolism. Methods Each preparation consisted of a NaCl solution, anhydrous milk fat labelled with a ¹³C mixed triacylglycerol, casein (as native phosphocaseinate powder with some lactose), and dipalmitoylphosphatidylcholine. Milk fat was either fed (i) unemulsified consecutively to the aqueous phase, or emulsified as (ii) coarse droplets of ∼10 μm covered mainly with the phospholipid, or (iii–iv) fine droplets of ∼1 μm covered mainly with casein, force–fed either in the liquid state or in a semi–crystallized state. ¹³C abundance in expired air samples was measured by isotope ratio mass spectrometry; results were expressed as ¹³C enrichment and were submitted to an ANOVA analysis. Results The ¹³CO₂ excretion curves of the unemulsified preparation and the coarse emulsion were similar and presented a sharp peak, both significantly different from the fine emulsion curves characterized by a nearly linear cumulative recovery. The crystalline state of the fine emulsion droplets and the viscosity of these emulsions did not affect significantly their excretion curves. The lipid metabolization (indicated by the ¹³C recovery) was significantly slower for the fine droplets coated with casein than for the large droplets coated with the phospholipid and the unemulsified fat. For the latter, a single ¹³C peak rapidly appeared, while for small droplets coated with caseins, ¹³C excretion was continuous up to 6 h. Conclusions Global lipid metabolism based on oxidation to CO₂ was decreased with smaller compared to larger emulsified milk fat particles with different coatings. These data support the concept that dairy products with different fat suprastructures are digested and metabolized differently.     

Substitution of casein by beta-casein or of whey protein isolate by alpha-lactalbumin does not affect mineral balance in growing rats

Bovine milk protein fractions that enable modification of the protein composition and amino acid profile of infant formulas to mimic those of human milk have recently become available. To determine the effects on protein quality and mineral bioavailability of replacing casein by beta-casein and of whey protein isolate by alpha-lactalbumin, 4 groups of growing rats were fed for 3 wk diets containing 10% protein as 1) casein (control); 2) beta-casein; 3) casein:whey (40:60); or 4) beta-casein:alpha-lactalbumin (40:60). Protein quality, determined as protein efficiency ratio (PER), net protein utilization (NPU), biological value (BV) and protein digestibility (PD), as well as body weight gain, were higher (P < 0.05) with consumption of the whey-adapted diets [casein:whey (40:60); beta-casein:alpha-lactalbumin (40:60)] compared with the casein diets (casein; beta-casein); however, there were no differences between the 2 casein diets or between the 2 whey-adapted diets. Apparent absorption of minerals (Ca, P, Fe, Zn) from the whey-adapted diets was higher than that from the casein diets (P < 0.05); but again, no differences were observed when casein or whey protein isolate were replaced by beta-casein or alpha-lactalbumin, respectively. Thus, substitution of casein by beta-casein or of whey protein isolate by alpha-lactalbumin does not affect protein quality or mineral bioavailability as determined in growing rats.     

Comparative Study of Cadmium Transfer in Ewe and Cow Milks During Rennet and Lactic Curds Preparation

Cadmium transfer from whole milk to cream, rennet, or lactic curds was studied before and following a repeated oral cadmium administration to three lactating ewes and one cow. Before cadmium administration, the cadmium levels in milk were around 0.4 μg/L in ewes and less than 0.2 μg/L in cow. Throughout cadmium administration the mean cadmium levels in milk were 3.3 ± 1.4 μg/L in ewes and 2.5 ± 1 μg/L in cow. During cadmium administration, 86% of cadmium in ewe milk was dispersed in the skimmed milk and 17% in the cream, whereas only 72% was dispersed in the cow skimmed milk and 27% in the cow cream. Most of milk cadmium was associated with casein fractions. About 70% of milk cadmium was transferred to the rennet or lactic curds of ewe. The remaining cadmium present in whole milk, about 9%, was transferred to the rennet or lactic curd whey. In cow, the proportion of cadmium associated with rennet or lactic curds, rennet curd whey, and lactic curd whey was, respectively, 60%, 56%, 14% and 12% of total milk cadmium. The fraction of total cadmium transferred from milk to its milk products, whatever the species, ranged from 94% to 103%. The factor of concentration of cadmium from whole milk to milk products ranged from three to six. We suggest that the excretion of cadmium into milk is mainly achieved via the milk casein secretion. This is, to our knowledge, the first in vivo study where the cadmium transfer from milk to its milk products after repeated cadmium oral administration to ewe and cow has been studied. Received: 27 October 1998/Accepted: 1 April 1999     

Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats

The aim of the work was to resolve whether glutamine and arginine supplemented diets affect plasma and tissue (muscle, liver and intestinal mucosa) glutamine concentrations, as well as glutaminase and glutamine synthetase specific activities. The trial was performed in growing rats fed 10% protein diets for 3 weeks. Protein sources were: whey proteins (W); whey proteins+free glutamine (WG); whey proteins+arginine (WA); and casein+wheat protein hydrolysate+acid whey (39:39:22), as source containing protein-bound glutamine (CGW). Rats fed the control diet (6.4% glutamine) (W) showed comparable glutamine body stores to those of rats fed the WG diet. In fact, glutamine sup- plementation down-regulated the hepatic glutamine synthetic capacity of growing rats (W/WG: 6.8+/-0.3 vs 6.0+/-0.2 nmol/min/mg protein). Arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease in plasma and tissue glutamine concentrations (W/WA: plasma, 1218+/-51 vs 1031+/-48 micromol/L; liver 7.5+/-0.4 vs 6.5+/-0.2 micromol/g; muscle: 5.7+/-0.2 vs 4.0+/-0.2 micromol/g). These data suggest that glutamine supplementation of the diet does not increase plasma and tissue glutamine concentrations in healthy growing rats, while the addition of arginine to the diet decreases glutamine body stores.     

Dietary whey protein increases liver and skeletal muscle glycogen levels in exercise-trained rats

We investigated the effect of different types of dietary protein on glycogen content in liver and skeletal muscle of exercise-trained rats. Twenty-four male Sprague-Dawley rats (approximately 100 g; n 6 per group) were divided into sedentary or exercise-trained groups with each group being fed either casein or whey protein as the source of dietary protein. Rats in the exercised groups were trained during 2 weeks using swimming exercise for 120 min/d, 6 d/week. Exercise training resulted in an increase in the skeletal muscle glycogen content. Furthermore, the whey protein group significantly increased the skeletal muscle glycogen content compared with the casein group. The increase in glycogen content in liver was significantly greater in rats fed the whey protein diet compared with those fed the casein diet. We also found that the whey protein diet increased the activity of liver glucokinase, whereas it decreased the activities of 6-phosphofructokinase and pyruvate kinase compared with the casein diet. However, hepatic total glycogen synthase activity and mRNA expression were similar with the two diets. In the skeletal muscle, whey protein decreased only 6-phosphofructokinase activity compared with casein. Total glycogen synthase activity in the skeletal muscle in the whey protein group was significantly higher than that in the casein group. The present study is the first to demonstrate that a diet based on whey protein may increase glycogen content in liver and skeletal muscle of exercise-trained rats. We also observed that whey protein regulated glycogen metabolism in these two tissues by different mechanisms.     

Dependence of the carbon-isotope contents of breath carbon dioxide, milk, serum and rumen fermentation products on the delta 13C value of food in dairy cows

Six dairy cows of two breeds were fed during three alternating periods with products from C3- and C4- plants to yield different natural 13C enrichments of the diet (delta 13C range: -28.0 to -13.7%). The resulting changes in the 13C enrichment of breath carbon dioxide, serum and milk of the animals followed the 13C:12C of the food, in agreement with the individual biological half-lives of those products, and established isotope discriminations. Breath CO2 was more enriched in 13C than expected. This could be related to isotope discriminations during rumen fermentation. From these results an isotopic balance model for the breath CO2 could be established.     

Phenylalanine kinetics differ between formula-fed and human milk-fed preterm infants

Infants fed casein-dominant formulas have higher plasma phenylalanine and tyrosine concentrations than those fed mother's milk. Conversely, elevated plasma threonine concentrations are observed in infants fed whey-dominant formulas. We recently showed that formula-fed preterm infants have a lower capacity to degrade threonine than do preterm infants fed mother's milk. We hypothesized that these same infants (n = 18) would differ in their catabolism of phenylalanine in response to phenylalanine loads provided by formulas with increasing casein content of formulas (whey:casein 60:40, 40:60, and 20:80) compared with preterm infants fed mother's milk. Plasma phenylalanine concentrations significantly rose (49, 46, 79 micromol . L(-1) for whey:casein 60:40, 40:60, and 20:80, respectively, pooled SD 8, P < 0.05); and plasma phenylalanine concentrations in infants fed mother's milk were low (40 +/- 4 micromol . L(-1)). Using [1-(13)C]phenylalanine tracer and (13)CO(2) production in breath we found that although there was a significant positive relation between phenylalanine oxidation and phenylalanine intake in formula-fed infants (r(2) = 0.43, P = 0.03), these infants were not able to increase their oxidation of phenylalanine enough to prevent a significant rise in plasma phenylalanine when fed the 20:80 formula. Compared to infants fed mother's milk, formula-fed infants had significantly lower phenylalanine oxidation (39.1 vs. 30.7% of phenylalanine intake, respectively, P < 0.05). We conclude that one of the mechanisms for the differences in plasma amino acid concentration between formula-fed and mother's milk-fed preterm infants may be in vivo down-regulated catabolism of 2 important essential amino acids (phenylalanine in addition to threonine) in formula-fed preterm infants.     

Unequal distribution of a stable isotopic calcium tracer between casein and whey fractions of infant formulas, human milk and cow's milk

Measurement of calcium absorption with tracers assumes a complete equilibration of tracer with milk calcium. In this study, the equilibration of tracer between the micellar casein and soluble fractions (primarily whey) of infant formulas, human milk and cow's milk was measured in vitro using milk samples enriched with 42Ca and analyzed by thermal ionization mass spectrometry. Incomplete equilibration of tracer occurred with the micellar casein fraction of all milks. The least equilibration with micellar casein was found with premature infant formula, for which the ratio of slopes of the equilibration lines (whey/casein) was 8.5/1. These differences may be due to Ca-casein binding in cow's milk-based formulas. The effects of the lack of tracer equilibration in vivo cannot be determined. However, unequal bioavailability of casein- vs. whey-bound Ca may exist.     

进食状态下口服~(13)C-亮氨酸呼出气平台期研究

目的探讨在进食状态下口服13C-亮氨酸作为示踪剂进行氨基酸代谢动力学研究时,呼出气中同位素含量在什么时间达到稳定的平台期。方法选取6名健康成年女性作为研究对象,给予日常膳食5天,在第6天持续口服13C-亮氨酸4h,并伴随食物摄入,收集给予同位素过程中及给予结束后1h的呼出气样本,测定13C的千分差值,以确定同位素的平台期。结果6名受试者的呼出气样品中13C的千分差值在口服同位素结束前后各半小时内处于相对较高的平台期,随后逐渐下降。结论在进食状态下口服13C-亮氨酸示踪剂进行氨基酸代谢动力学研究,取样的适宜时间是在给予同位素结束前后各半小时内。     

Whey protein isolate and glycomacropeptide decrease weight gain and alter body composition in male Wistar rats

The effect of feed protein type on body composition and growth has been examined. Evidence exists that whey protein concentrate is effective at limiting body fat expansion. The presence of caseinomacropeptide, a mixture of glycosylated and non-glycosylated carbohydrate residues, in particular glycomacropeptide (GMP) in whey protein concentrate may be important for this effect. The influence of whey protein isolate (WPI) and GMP on weight gain and body composition was examined by feeding Wistar rats ad libitum for 7 weeks with five semi-purified American Institute of Nutrition-based diets differing in protein type: (1) casein; (2) barbequed beef; (3) control WPI (no GMP); (4) WPI+GMP at 100 g/kg; (5) WPI+GMP at 200 g/kg. Body composition was assessed, and plasma samples were assayed for TAG, insulin and glucose. Body-weight gain was lower (- 21 %) on the control WPI diet relative to casein, with a non-significant influence associated with GMP inclusion (- 30 %), the effect being equivalent at both levels of GMP addition. Renal and carcass fat mass were reduced in the highest GMP diet when compared with WPI (P < 0.05). Plasma insulin was lowered by GMP at the highest addition compared with WPI alone (- 53 %; P < 0.01). Plasma TAG in the WPI+GMP (200 g/kg) group were lower (- 27 %; P < 0.05) than the casein and beef groups. In conclusion, GMP appears to have a significant additional influence when combined with WPI on fat accumulation. WPI alone appears to have the predominant influence accounting for 70 % of the overall effect on body-weight gain. Mechanisms for this effect have not been identified but food intake was not responsible.     

Rate of endogenous methionine oxidation in rats at different levels of methionine intake

The fractional rates of loss of 14C and body-weight were measured in growing female rats after a single dose of [l-14C]methionine. After the dose, the rats were maintained for 30 d on diets containing graded amounts of methionine and killed at 30 d. By 20 d after the dose the level of labelling of body proteins was fairly uniform. From 20 to 30 d, 14CO2 was collected at intervals. The rats were killed at 30 d and the total radioactivity remaining in the body was measured. The fractional loss of 14CO2 between days 20 and 30 was constant regardless of methionine intake except at the highest intake level. After correcting the fractional loss of 14CO2 for urinary 14C loss, a value of 35 mg/kg body-weight (20 mg/kg body-weight 0.75) was obtained for the rate of endogenous methionine oxidation.     

Dietary interactions influence the effects of bovine folate-binding protein on the bioavailability of tetrahydrofolates in rats

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.     

13C-ethanol and 13C-acetate breath tests in normal and aldehyde dehydrogenase deficient individuals

Four normal and five aldehyde dehydrogenase (ALDH) isozyme I deficient individuals were subsequently loaded with (1-13C)ethanol and (1-13C)sodium acetate and the conversion of the label to 13CO2 was determined in expired air by isotope ratio mass spectrometry. In the 13C-acetate breath test, both groups showed virtually identical recovery of the label in expired air, namely 48.5 +/- 2.3% (mean +/- S.D.) for normal and 46.8 +/- 5.7% for deficient individuals. However, in the 13C-ethanol breath test, both the groups performed differently. On average, although a certain overlap of the single data was observed, the recovery of the label after four hours was 43.4 +/- 3.8% for the normal and 35.6 +/- 6.8% for the ALDH deficient subjects. These findings suggest a slower conversion of ethanol to carbon dioxide in aldehyde dehydrogenase deficient individuals, which may be another consequence of this deficiency besides the higher plasma acetaldehyde levels observed after ethanol loading in comparison to individuals with normal aldehyde dehydrogenase activity.     

Use of a ventilated canopy for assessment of [13C]leucine oxidation in patients receiving total parenteral nutrition

Rates of oxidation of infused 13C-labeled substrates are calculated from CO2 production and 13C enrichment in breath CO2. Breath sampling through a mouthpiece is not appropriate in severely ill patients; the authors therefore validated the use of direct air sampling from the ventilated canopy of an indirect calorimeter for measuring the oxidation of 13C-labeled substrates. Infusions of H13CO3Na or L-[1-13C]leucine were performed in four healthy postabsorptive adults and six malnourished patients receiving total parenteral nutrition (TPN). At each sampling point, air was collected from the canopy to compare with breath air sampled through a mouthpiece and 13CO2 enrichment determined by isotope ratio mass spectrometry. Despite five-fold dilution of expired air by room air within the canopy (a dilution required to maintain safe CO2 levels in inspired air): (1) Breath 13CO2 enrichment was accurately predicted using samples from the canopy, with a correction taking into account the measured CO2 fractions in canopy and room air; (2) the precision in isotopic determination was similar with both methods (SD/mean of 12 determinations = 2.5 +/- 1.0% vs 3.0 +/- 1.0%). These data demonstrate that the use of a ventilated canopy allows for combined assessment of energy expenditure and rates of oxidation of 13C-labeled substrates even in sick, debilitated patients receiving total parenteral nutrition.     

Substrate utilization during exercise performed with and without glucose ingestion in female and male endurance trained athletes

Compared to males, females oxidize proportionately more fat and less carbohydrate during endurance exercise performed in the fasted state. This study was designed to test the hypothesis that there may also be gender differences in exogenous carbohydrate (CHOexo) oxidation during exercise. Healthy, young males (n = 7) and females (n = 7) each completed 2 exercise trials (90 min cycle ergometry at 60% VO2peak), 1 week apart. Females were eumenorrheic and were tested in the midfollicular phase of their menstrual cycle. Subjects drank intermittently either 8% CHOexo (1 g glucose x kg x h(-1)) enriched with U-13C glucose or an artificially sweetened placebo during the trial. Whole-body substrate oxidation was determined from RER, urinary urea excretion, and the ratio of 13C:12C in expired gas during the final 60 min of exercise. During the placebo trial, fat oxidation was higher in females then in males (0.42 +/- 0.07 vs 0.32 +/- 0.09 g.min(-1).kg LBM(-1) x 10(2)) at 30 min of exercise (p < .05). When averaged over the final 60 min of exercise, the relative proportions of fat, total carbohydrate, and protein were all similar between groups. During CHOexo ingestion, both the ratio of 13C:12C in expired gas (p < .05) and the proportion of energy derived from CHOexo relative to LBM (p < .05) were higher in females compared to males at 75- and 90-min exercise. When averaged over the final 60 min of exercise, the percentage of CHOexo to the total energy contribution tended to be higher in females (14.3 +/- 1.2%) than in males (11.2 +/- 1.2%; p = .05). Compared to males, females may oxidize a greater relative proportion of CHOexo during endurance exercise which, in turn, may spare more endogenous fuel. Based on these observations, ingested carbohydrate may be a particularly beneficial source of fuel during endurance exercise for females.     

Compartmentalization and quantitation of protein in human milk

Human milk protein was determined by three colorimetric methods and by Kjeldahl analysis. The distribution of nitrogen (N) and protein was determined within various milk compartments. Total N, whey, casein, nonprotein nitrogen (NPN), cell N and N in the fat fraction were analyzed by micro-Kjeldahl analysis after a series of centrifugation and ultracentrifugation separations. Fresh milk samples (colostrum, transitional milk and mature milk) were centrifuged at 500 X g to separate milk cells and at 5000 X g to skim the milk. Decelled milk and skimmed milk were ultracentrifuged at 189,000 X g to separate fat and casein micelles from whey. NPN was analyzed after trichloroacetic acid precipitation. Whole milk, decelled milk, skimmed milk and whey were analyzed for protein with the Lowry method, modified for fat-containing samples, the Bradford dye-binding assay (Bio-Rad) and the Pierce bicinchoninic acid (BCA) assay. Cell nitrogen had a tendency to be lower in mature milk than in colostrum. Colostrum contained only 6% casein protein, whereas mature milk contained 13%. Fat from skimming was lower in N than fat from ultracentrifugation. Average NPN levels were similar for milk from all three lactation periods, and constituted 10% of colostrum N and 25% of mature milk N. Protein determined by the Bio-Rad method on whole milk samples had the lowest variability (square root MSE) when correlated to Kjeldahl values. All three assays had lower variability when analyzing whey and skimmed milk than when analyzing whole milk. The Lowry method and the Bio-Rad method had low variability for whey and skimmed milk samples, but the Lowry method yielded analytical values closest to Kjeldahl protein values. The BCA method consistently overestimated Kjeldahl protein by 30%.     

Utilization of zinc from picolinic or citric acid complexes in relation to dietary protein source in rats

Young male Sprague-Dawley rats were divided in 18 groups of eight animals each and were fed ad libitum for 24 d a purified diet with 20% casein, whey protein or serum albumin as protein source. Each diet was supplemented with zinc picolinate, zinc citrate or zinc sulfate to a level of dietary zinc equal to 5 or 10 ppm. The source of zinc had no significant effect on zinc utilization with any of the three dietary proteins or at either dietary zinc concentration. With the 5 ppm Zn diet zinc concentration in the serum, but not weight gain or the zinc concentration in femur, testis or whole body was lower in rats fed the casein diet than in those fed the whey diet. Zinc concentrations in serum, femur, testis and whole body, but not weight gain, were lower in rats fed the casein diet than in those fed the serum albumin diet. With the 10 ppm Zn diet, zinc concentrations in serum, femur and whole body, but not weight gain nor zinc concentration in testis, were lower in rats fed the casein diet than in those fed either of the other diets. These results suggest that the reason for the lower utilization of zinc from cow milk in comparison to human milk may be the higher casein concentration in cow milk.     

Effect of milk protein and fat intake on blood pressure and the incidence of cerebrovascular diseases in stroke-prone spontaneously hypertensive rats (SHRSP)

The intake of two milk protein-rich diets containing casein and whey protein attenuated the development of severe hypertension in stroke-prone spontaneously hypertensive rats (SHRSP), and extended their life span in comparison with SHRSP on a regular stock diet. Milk fat-rich diet intake reduced the incidence of cerebrovascular disease in SHRSP without a significant fall in blood pressure. These results suggest that certain milk components have a preventive effect on hypertension and cerebrovascular disease in SHRSP.