Warren D. Kruger

Reduction of Plasma Homocyst(e)ine Levels by Breakfast Cereal Fortified with Folic Acid in Patients with Coronary Heart Disease

BACKGROUND The Food and Drug Administration (FDA) has recommended that cereal-grain products be fortified with folic acid to prevent congenital neural-tube defects. Since folic acid supplementation reduces levels of plasma homocyst(e)ine, or plasma total homocysteine, which are frequently elevated in arterial occlusive disease, we hypothesized that folic acid fortification might reduce plasma homocyst(e)ine levels. METHODS To test this hypothesis, we assessed the effects of breakfast cereals fortified with three levels of folic acid, and also containing the recommended dietary allowances of vitamins B6 and B12, in a randomized, double-blind, placebo-controlled, crossover trial in 75 men and women with coronary artery disease. RESULTS Plasma folic acid increased and plasma homocyst(e)ine decreased proportionately with the folic acid content of the breakfast cereal. Cereal providing 127 microg of folic acid daily, approximating the increased daily intake that may result from the FDAs enrichment policy, increased plasma folic acid by 31 percent (P=0.045) but decreased plasma homocyst(e)ine by only 3.7 percent (P= 0.24). However, cereals providing 499 and 665 microg of folic acid daily increased plasma folic acid by 64.8 percent (P<0.001) and 105.7 percent (P=0.001), respectively, and decreased plasma homocyst(e)ine by 11.0 percent (P<0.001) and 14.0 percent (P=0.001), respectively. CONCLUSIONS Cereal fortified with folic acid has the potential to increase plasma folic acid levels and reduce plasma homocyst(e)ine levels. Further clinical trials are required to determine whether folic acid fortification may prevent vascular disease. Until then, our results suggest that folic acid fortification at levels higher than that recommended by the FDA may be warranted.

Cystathionine β‐synthase mutations in homocystinuria

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine β‐synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety‐two different disease‐associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine‐responsive I278T and the pyridoxine‐nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene. Hum Mutat 13:362–375, 1999.

Evidence for heme-mediated redox regulation of human cystathionine beta-synthase activity.

Human cystathionine β-synthase catalyzes the first step in the catabolic removal of the toxic metabolite, homocysteine. It is unique in being dependent on both pyridoxal phosphate (PLP) and heme for activity. The reaction involves condensation of serine and homocysteine to give cystathionine. Although the role of PLP can be rationalized in analogy with other PLP-dependent enzymes that catalyze β-replacement reactions, the role of the heme is unknown. In this study, we have purified and characterized the recombinant human enzyme and have examined the effect of heme oxidation state on enzyme activity. We find that under reducing conditions, generated by addition of titanium citrate, the enzyme exhibits a 1.7-fold lower activity than under oxidizing conditions. Reoxidation of the ferrous enzyme with ferricyanide results in alleviation of inhibition. This redox-linked change in enzyme activity correlates with changes in heme oxidation state monitored by UV-visible spectroscopy. Dithiothreitol, which does not reduce the enzyme-bound heme, does not perturb enzyme activity. These studies provide the first evidence for redox-linked regulation of cystathionine β-synthase which is heme-dependent.

Hyperhomocysteinemia Promotes Inflammatory Monocyte Generation and Accelerates Atherosclerosis in Transgenic Cystathionine β-Synthase–Deficient Mice

Background— Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. Methods and Results— We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine &bgr;-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE−/− Cbs−/− mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-&agr; and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE−/− Cbs−/− mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE−/− Cbs−/− mice and another severe HHcy mouse model (Tg-S466L Cbs−/−) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6Chi and Ly-6Cmid monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs−/− and Tg-hCBS apoE−/− Cbs−/− mice. These changes were exacerbated in Tg-S466L Cbs−/− mice with aging. Addition of l-homocysteine (100 to 500 &mgr;mol/L), but not l-cysteine, maintained the Ly-6Chi subset and induced the Ly-6Cmid subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6Cmid subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. Conclusion— HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase–mediated oxidant stress.

Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice.

Genetic disorders of homocysteine (Hcy) or folate metabolism or high‐methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy‐thiolac‐tone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine ς‐synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy‐thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (A‐Hcy‐protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how A–Hcy‐protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma A‐Hcy‐protein was elevated 10‐fold in mice fed a high‐methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton‐coupled folate transporter (Pcft) gene resulted in a 10‐to 30‐fold increase in plasma or serum AHcy‐protein levels. Liver AHcy‐protein was elevated 3.4‐fold in severely and 11‐fold in extremely hyperhomocysteinemic Cbs‐deficient mice, 3.6‐fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr‐deficient animals, suggesting that mice have a capacity to prevent accumulation of A‐Hcy‐protein in their organs. These findings provide evidence that A‐Hcy‐protein is an important metabolite associated with Hcy pathophysiology in the mouse.—Jakubowski, H.,Peria‐Kajan, J., Finnell, R.H., Cabrera, R.M., Wang, H., Gupta, S., Kruger, W.D., Kraus, J.P., Shih, D.M. Genetic or nutritional disorders in homocysteine or folate metabolism increase protein A‐homocysteinylation in mice. FASEB J. 23, 1721–1727 (2009)

Reduction of homocysteine levels in coronary artery disease by low-dose folic acid combined with vitamins B6 and B12 ☆

An increased plasma homocysteine concentration is a risk factor for atherosclerosis. Folic acid lowers homocysteine but the optimal dose in patients with coronary artery disease (CAD) is unclear. This placebo-controlled, single-blind, dose-ranging study evaluates the effect of low-dose folic acid on homocysteine levels in 95 patients aged 61 +/- 11 years (mean +/- SD) with documented CAD. Patients in each group were given either placebo or 1 of 3 daily supplements of folic acid (400 microg, 1 mg, or 5 mg) for 3 months. Each active treatment arm also received 500 microg vitamin B12 and 12.5 mg vitamin B6. Total plasma homocysteine levels were measured after 30 and 90 days. Folic acid 400 microg reduced homocysteine levels from 13.8 +/- 8.8 to 9.6 +/- 2.0 micromol/L at 90 days (p = 0.001). On 1- and 5-mg folic acid, levels decreased from 13.0 +/- 6.4 to 9.8 +/- 4.0 micromol/L (p = 0.001) and from 14.8 +/- 6.9 to 9.7 +/- 3.3 micromol/L (p < 0.001), respectively. The decrease was similar in all treatment groups. There was no significant change with placebo. Although the sample size is small, these findings suggest that daily administration of 400 microg/day folic acid combined with vitamin B12 and vitamin B6 may be equivalent to higher doses in reducing homocysteine levels in patients with CAD.

Modulation of Cystathionine β-Synthase Level Regulates Total Serum Homocysteine in Mice

Elevated total plasma homocysteine is an independent risk factor in the development of vascular disease in humans. Cystathionine β-synthase (CBS) is an enzyme that condenses homocysteine with serine to form cystathionine. In this article, we describe the effects of modulating CBS activity using a transgenic mouse that contains the human CBS cDNA under control of the zinc-inducible metallothionein promoter (Tg–CBS). In the presence of zinc, Tg–CBS mice have a 2- to 4-fold increase in liver and kidney CBS activity compared with nontransgenic littermates. Transgenic mice on standard mouse chow had a 45% decrease in their serum homocysteine (12.1 to 7.2 μmol/L; P<0.0001) when zinc was added to drinking water, although zinc had minimal effect on their nontransgenic siblings (13.2 μmol/L versus 13.0 μmol/L; P=NS). Tg–CBS mice maintained on a high-methionine, low-folate diet also had significantly lower serum homocysteine compared with control animals (179 μmol/L versus 242 μmol/L; P<0.02). CBS overexpression also significantly lowered serum cysteinylglycine (3.6 versus 2.8 μmol/L; P<0.003) levels and reduced the levels of many amino acids in the liver. We also found that expression of Tg-CBS rescued the severe hyperhomocysteinemia and neonatal lethality of Cbs deletion animals. Our results show that elevating CBS activity is an effective method to lower plasma homocysteine levels. In addition, the creation of an inducible mouse system to modulate plasma homocysteine will also be useful in the study of homocysteine-related vascular disease.

Tsc1+ and tsc2+ regulate arginine uptake and metabolism in Schizosaccharomyces pombe

Mutations in either TSC1 or TSC2 cause tuberous sclerosis complex, an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the skin, brain, heart, and kidneys. Homologs for the TSC1 and TSC2 genes have been identified in mouse, rat, Fugu, Drosophila, and in the yeast Schizosaccharomyces pombe. Here we show that S. pombe lacking tsc1+ or tsc2+ have similar phenotypes including decreased arginine uptake, decreased expression of three amino acid permeases, and low intracellular levels of four members of the arginine biosynthesis pathway. Recently, the small GTPase Rheb was identified as a target of the GTPase-activating domain of tuberin in mammalian cells and in Drosophila. We show that the defect in arginine uptake in cells lacking tsc2+ is rescued by the expression of a dominant negative form of rhb1+, the Rheb homolog in S. pombe, but not by expressing wild-type rhb1+. Expression of the tsc2+ gene with a patient-derived mutation within the GAP domain did not rescue the arginine uptake defect in tsc2+ mutant yeast. Taken together, these findings support a model in which arginine uptake is regulated through tsc1+, tsc2+, and rhb1+ in S. pombe and also suggest a role for the Tsc1 and Tsc2 proteins in amino acid biosynthesis and sensing.

The Birt-Hogg-Dube and Tuberous Sclerosis Complex Homologs Have Opposing Roles in Amino Acid Homeostasis in Schizosaccharomyces pombe

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Δtsc1 and Δtsc2, were up-regulated in Δbhd, and levels of specific intracellular amino acids known to be low in Δtsc1 and Δtsc2 were elevated in Δbhd. This “opposite” profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1+ dramatically increased permease expression levels in Δbhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Δbhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.

Mouse models of cystathionine β-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia

Untreated cystathionine β‐synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 µΜ), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 µΜ). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg‐hCBS Cbs–/– mice, with a mean serum tHcy of 169 µΜ, and Tg‐I278T Cbs–/– mice, with a mean tHcy of 296 µΜ. Only Tg‐I278T Cbs–/– animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg‐I278T Cbs–/– mice have significantly elevated levels of free oxidized homocysteine but not protein‐bound homocysteine in serum and elevation of all forms of homocysteine and S‐adenosyl‐homocysteine in the liver compared to Tg‐hCBS Cbs–/– mice. RNA profiling of livers indicate that Tg‐I278T Cbs–/– and Tg‐hCBS Cbs–/– mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.— Gupta, S., Kuhnisch, J., Mustafa, A., Lhotak, S., Schlachterman, A., Slifker, M. J., Klein‐Szanto, A., High, K. A., Austin, R. C., Kruger, W. D. Mouse models of cystathionine β‐synthase deficiency reveal significant threshold effects of hyperhomocysteinemia. FASEB J. 23, 883–893 (2009)