Meal cysteine improves postprandial glucose control in rats fed a high-sucrose meal.

Whey protein, particularly the alpha-lactalbumin fraction, are rich in cysteine (cys) and could therefore favor postprandial glucose homeostasis by a glutathione-mediated effect. This work investigates the effects of the ingestion of an alpha-lactalbumin-rich whey concentrate (alpha-LAC) during a high-sucrose (HS) meal on postprandial glucose homeostasis in healthy rats. In the first experiment, rats received an HS meal containing 14% protein, in which the protein source was either alpha-LAC (HS(a)) or total milk proteins, alone (HS(0)) or supplemented with 17 mg (HS(1)) or 59 mg (HS(2)) of N-acetylcysteine (NAC). This resulted in a total cys content 3.6-fold higher in the HS(1) and HS(a) meals and 12-fold higher in the HS(2) meal, when compared to the HS(0) meal. Postprandial parameters were monitored for 3 h after ingestion of the meal. The same measurements were performed on rats injected with 4 mmol/kg of buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Increasing the meal's cys content dose-dependently reduced both postprandial glucose and insulin (P<.05). The inhibition of glutathione synthesis with BSO injection abrogated the beneficial effects of NAC supplementation on postprandial glucose response but did not affect those of alpha-LAC. These results show that (1) the substitution of alpha-LAC for total milk protein reduces glucose response, as does the addition of a cys donor to the meal, (2) but contrary to those of a simple cys donor, the beneficial effects of alpha-LAC are not entirely mediated by glutathione synthesis, suggesting additional mechanisms.

Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line.

BACKGROUND: Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable. PATIENTS AND METHODS: The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose L-PAM. RESULTS: CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0-1,000 micromol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to L-PAM (LD90 = 42 micromol/L; peak plasma concentration in the patient equals 3.9 micromol/L) and moderately resistant to BSO (LD90 = 509 micromol/L; steady-state concentration in the patient equals 397 micromol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3-4 logs of cell kill, combination index <1) at clinically achievable levels of BSO (100-400 micromol/L) and levels of L-PAM (10-40 micromol/L) clinically achievable only with hematopoietic stem cell support. CONCLUSIONS: The in vitro results obtained for CHLA-171 suggest that BSO/L-PAM therapy may be optimally effective for drug-resistant neuroblastoma using myeloablative doses of L-PAM.

Effect of thiol status on nitric oxide metabolism in the circulation.

To elucidate the dynamics of nitric oxide (NO) metabolism in the circulation and its relationship with glutathione metabolism, formation of nitrosylhemoglobin (NO-Hb), S-nitrosothiols (RSNO), and nitrite+nitrate (NOx) was determined in blood samples from normal rats and animals that were treated with a loading dose of GSH or L-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. When incubated in vitro with 0.2 mM NOC7, an NO donor, NO-Hb levels increased rapidly, peaked at 10 min, and decreased thereafter with a half-life of 35 min in blood samples from control, BSO-treated, or GSH-loaded animals. Levels of low-molecular-weight RSNO in plasma samples from the three animal groups also increased transiently, peaked at 10 min, and decreased thereafter. However, the amount of RSNO formed in GSH-loaded rat plasma was significantly greater than in control and BSO-treated animals. Plasma levels of NOx rapidly and similarly increased in all animal groups. Intravenously injected NOC7 also generated NO-Hb in circulating erythrocytes. In control animals, blood levels of NO-Hb increased maximally at 30 min and decreased thereafter with a half-life of 100 min. NO-Hb formed in the GSH-loaded group was significantly lower than in the control group. In contrast, the rate of NO-Hb formation was significantly higher with the BSO-treated group than with the control group. Although NOC7 did not affect the plasma levels of low-molecular-weight RSNO in plasma of both control and BSO-treated groups, it significantly increased RSNO in the GSH-loaded group. Thirty minutes after administration of NOC7, about 20% of the dose was recovered as plasma NOx in all animal groups. These results suggested that GSH status in animals might affect the metabolism of NO.